Bio

Bio


Research interests in Khosla's laboratory lie at the interface of enzyme chemistry and medicine. For the past several years, we have investigated the catalytic mechanisms of modular megasynthases such as polyketide synthases, with the concomitant goal of harnessing their programmable chemistry for preparing pharmaceutically relevant natural products. Recent accomplishments include methods for heterologous production of polyketides; genetically reprogrammed biosynthesis of anthraquinones and polypropionates; and chemo-biosynthesis of new polyketides not readily affordable by synthetic or biological methods alone. These methodologies are already finding practical use. At the same time, we have placed a major emphasis on the biochemistry and structural biology of these giant protein assemblies. Fundamental insights into assembly line biosynthetic mechanisms have emerged, including the finding that protein-protein interactions play a central role in intermodular communications. In turn, these insights are highlighting opportunities for enhancing the efficiency of biosynthetic engineering. Over the next decade we envision that the predictive power of polyketide biosynthetic engineering will mature analogous to current protein engineering capabilities.

More recently, we have investigated the pathogenesis of Celiac Sprue, an HLA-DQ2 associated inflammatory disease of the small intestine that is induced by exposure to gluten from foodgrains such as wheat, rye and barley. Within the past few years, we have explored three potential therapeutic strategies for this widespread but overlooked disease. By dissecting the unique chemical features of gluten, we discovered an intimate link between proteolytic stability and immunotoxicity of gluten, and translated this knowledge into the design of an oral enzyme therapy for the disease. We have also synthesized and evaluated mechanism-based inhibitors of human transglutaminase 2, the predominant disease associated auto-antigen, and used them to deduce the high-resolution structure of the antigen-bound protein. Finally, our structural and mechanistic dissection of HLA-DQ2 has been used to design, synthesize and evaluate gluten peptide analogues that selectively inhibit disease associated T cells. We remain committed to the vision that, within the next decade, safe and effective drugs will start having measurable impact on the health of celiac sprue patients.

Academic Appointments


Honors & Awards


  • Member, National Academy of Engineering (2009)
  • Arthur C. Cope Scholar Award, American Chemical Society (2009)
  • Member, American Academy of Arts and Sciences (2007)
  • Fellow, American Association for the Advancement of Science (2006)
  • Pure Chemistry Award, American Chemical Society (2000)
  • Eli Lilly Award in Biological Chemistry, American Chemical Society (1999)
  • Alan T Waterman Award, National Science Foundation (1999)

Boards, Advisory Committees, Professional Organizations


  • Member, National Academy of Engineering (2013 - Present)
  • Member, American Academy of Arts and Sciences (2013 - Present)

Professional Education


  • PhD, California Institute of Technology (1990)

Research & Scholarship

Current Research and Scholarly Interests


Research interests in this laboratory lie at the interface of chemistry and medicine.

For the past several years, we have investigated the catalytic mechanisms of modular megasynthases such as polyketide synthases, with the concomitant goal of harnessing their programmable chemistry for preparing new antibiotics. Recent accomplishments include methods for heterologous production of polyketides; genetically reprogrammed biosynthesis of anthraquinones and polypropionates; and chemo-biosynthesis of new polyketides not readily affordable by synthetic or biological methods alone. These methodologies are already finding practical use. At the same time, we have placed a major emphasis on the biochemistry and structural biology of these giant protein assemblies. Fundamental insights into assembly line biosynthetic mechanisms have emerged, including the finding that protein-protein interactions play a central role in intermodular communications. In turn, these insights are highlighting opportunities for enhancing the efficiency of biosynthetic engineering. Over the next decade we envision that the predictive power of polyketide biosynthetic engineering will mature analogous to current protein engineering capabilities.

More recently, we have investigated the pathogenesis of celiac sprue, an HLA-DQ2 associated autoimmune disease of the small intestine that is induced by exposure to gluten from foodgrains such as wheat, rye and barley. Within the past few years, we have explored three potential therapeutic strategies for this widespread but overlooked disease. By dissecting the unique chemical features of gluten, we discovered an intimate link between proteolytic stability and immunotoxicity of gluten, and translated this knowledge into the design of an oral enzyme therapy for the disease. At the same time, we have synthesized and evaluated mechanism-based inhibitors of human transglutaminase 2, the predominant disease associated auto-antigen. Finally, our structural and mechanistic dissection of HLA-DQ2 has been used to design, synthesize and evaluate gluten peptide analogues that selectively inhibit disease associated T cells. We remain committed to the vision that, within the next decade, safe and effective drugs will start having measurable impact on the health of celiac sprue patients.

Teaching

2013-14 Courses


Graduate and Fellowship Programs


Publications

Journal Articles


  • Stereochemistry of Reductions Catalyzed by Methyl-Epimerizing Ketoreductase Domains of Polyketide Synthases JOURNAL OF THE AMERICAN CHEMICAL SOCIETY You, Y., Khosla, C., Cane, D. E. 2013; 135 (20): 7406-7409

    Abstract

    Ketoreductase (KR) domains from modular polyketide synthases (PKSs) catalyze the reduction of 2-methyl-3-ketoacyl acyl carrier protein (ACP) substrates and in certain cases epimerization of the 2-methyl group as well. The structural and mechanistic basis of epimerization is poorly understood, and only a small number of such KRs been studied. In this work, we studied three recombinant KR domains with putative epimerase activity: NysKR1 from module 1 of the nystatin PKS, whose stereospecificity can be predicted from both the protein sequence and the product structure; RifKR7 from module 7 of the rifamycin PKS, whose stereospecificity cannot be predicted from the protein sequence; and RifKR10 from module 10 of the rifamycin PKS, whose specificity is unclear from both the sequence and the structure. Each KR was individually incubated with NADPH and (2R)- or (2RS)-2-methyl-3-ketopentanoyl-ACP generated enzymatically in situ or via chemoenzymatic synthesis, respectively. Chiral GC-MS analysis revealed that each KR stereospecifically produced the corresponding (2S,3S)-2-methyl-3-hydroxypentanoyl-ACP in which the 2-methyl substituent had undergone KR-catalyzed epimerization. Thus, our results have led to the identification of a prototypical set of KR domains that generate (2S,3S)-2-methyl-3-hydroxyacyl products in the course of polyketide biosynthesis.

    View details for DOI 10.1021/ja4014776

    View details for Web of Science ID 000319551000005

    View details for PubMedID 23659177

  • Gluten-sensitive enteropathy coincides with decreased capability of intestinal T cells to secrete IL-17 and IL-22 in a macaque model for celiac disease CLINICAL IMMUNOLOGY Xu, H., Feely, S. L., Wang, X., Liu, D. X., Borda, J. T., Dufour, J., Li, W., Aye, P. P., Doxiadis, G. G., Khosla, C., Veazey, R. S., Sestak, K. 2013; 147 (1): 40-49

    Abstract

    Celiac disease (CD) is an autoimmune disorder caused by intolerance to dietary gluten. The interleukin (IL)-17 and IL-22 function as innate regulators of mucosal integrity. Impaired but not well-understood kinetics of the IL-17/22 secretion was described in celiac patients. Here, the IL-17 and IL-22-producing intestinal cells were studied upon their in vitro stimulation with mitogens in class II major histocompatibility complex-defined, gluten-sensitive rhesus macaques. Pediatric biopsies were collected from distal duodenum during the stages of disease remission and relapse. Regardless of dietary gluten content, IL-17 and IL-22-producing cells consisted of CD4+ and CD8+ T lymphocytes as well as of lineage-negative (Lin-) cells. Upon introduction of dietary gluten, capability of intestinal T cells to secrete IL-17/22 started to decline (p<0.05), which was paralleled with gradual disruption of epithelial integrity. These data indicate that IL-17/22-producing cells play an important role in maintenance of intestinal mucosa in gluten-sensitive primates.

    View details for DOI 10.1016/j.clim.2013.02.012

    View details for Web of Science ID 000317810200007

    View details for PubMedID 23518597

  • Mechanism and Specificity of an Acyltransferase Domain from a Modular Polyketide Synthase BIOCHEMISTRY Dunn, B. J., Cane, D. E., Khosla, C. 2013; 52 (11): 1839-1841

    Abstract

    Acyltransferase (AT) domains of modular polyketide synthases exercise tight control over the choice of ?-carboxyacyl-CoA substrates, but the mechanistic basis for this specificity is unknown. We show that whereas the specificity for the electrophilic malonyl or methylmalonyl component is primarily expressed in the first half-reaction (formation of the acyl-enzyme intermediate), the second half-reaction shows comparable specificity for the acyl carrier protein that carries the nucleophilic pantetheine arm. We also show that currently used approaches for engineering AT domain specificity work mainly by degrading specificity for the natural substrate rather than by enhancing specificity for alternative substrates.

    View details for DOI 10.1021/bi400185v

    View details for Web of Science ID 000316520000001

    View details for PubMedID 23452124

  • Analysis and Refactoring of the A-74528 Biosynthetic Pathway JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Fitzgerald, J. T., Charkoudian, L. K., Watts, K. R., Khosla, C. 2013; 135 (10): 3752-3755

    Abstract

    A-74528 is a C30 polyketide natural product that functions as an inhibitor of 2',5'-oligoadenylate phosphodiesterase (2'-PDE), a key regulatory enzyme of the interferon pathway. Modulation of 2'-PDE represents a unique therapeutic approach for regulating viral infections. The gene cluster responsible for biosynthesis of A-74528 yields minute amounts of this natural product together with considerably larger quantities of a structurally dissimilar C30 cytotoxic agent, fredericamycin. Through construction and analysis of a series of knockout mutants, we identified the genes necessary for A-74528 biosynthesis. Remarkably, the formation of six stereocenters and the regiospecific formation of six rings in A-74528 appear to be catalyzed by only two tailoring enzymes, a cyclase and an oxygenase, in addition to the core polyketide synthase. The inferred pathway was genetically refactored in a heterologous host, Streptomyces coelicolor CH999, to produce 3 mg/L A-74528 in the absence of fredericamycin.

    View details for DOI 10.1021/ja311579s

    View details for Web of Science ID 000316244200006

    View details for PubMedID 23442197

  • Selective Inhibition of Extracellular Thioredoxin by Asymmetric Disulfides JOURNAL OF MEDICINAL CHEMISTRY DiRaimondo, T. R., Plugis, N. M., Jin, X., Khosla, C. 2013; 56 (3): 1301-1310

    Abstract

    Whereas the role of mammalian thioredoxin (Trx) as an intracellular protein cofactor is widely appreciated, its function in the extracellular environment is not well-understood. Only few extracellular targets of Trx-mediated thiol-disulfide exchange are known. For example, Trx activates extracellular transglutaminase 2 (TG2) via reduction of an intramolecular disulfide bond. Because hyperactive TG2 is thought to play a role in various diseases, understanding the biological role of extracellular Trx may provide critical insight into the pathogenesis of these disorders. Starting from a clinical-stage asymmetric disulfide lead, we have identified analogs with >100-fold specificity for Trx. Structure-activity relationship and computational docking model analyses have provided insights into the features important for enhancing potency and specificity. The most active compound identified had an IC(50) below 0.1 ?M in cell culture and may be appropriate for in vivo use to interrogate the role of extracellular Trx in health and disease.

    View details for DOI 10.1021/jm301775s

    View details for Web of Science ID 000315182100055

    View details for PubMedID 23327656

  • Engineering the acyltransferase substrate specificity of assembly line polyketide synthases. Journal of the Royal Society, Interface / the Royal Society Dunn, B. J., Khosla, C. 2013; 10 (85): 20130297-?

    Abstract

    Polyketide natural products act as a broad range of therapeutics, including antibiotics, immunosuppressants and anti-cancer agents. This therapeutic diversity stems from the structural diversity of these small molecules, many of which are produced in an assembly line manner by modular polyketide synthases. The acyltransferase (AT) domains of these megasynthases are responsible for selection and incorporation of simple monomeric building blocks, and are thus responsible for a large amount of the resulting polyketide structural diversity. The substrate specificity of these domains is often targeted for engineering in the generation of novel, therapeutically active natural products. This review outlines recent developments that can be used in the successful engineering of these domains, including AT sequence and structural data, mechanistic insights and the production of a diverse pool of extender units. It also provides an overview of previous AT domain engineering attempts, and concludes with proposed engineering approaches that take advantage of current knowledge. These approaches may lead to successful production of biologically active 'unnatural' natural products.

    View details for DOI 10.1098/rsif.2013.0297

    View details for PubMedID 23720536

  • Regulation of the activities of the mammalian transglutaminase family of enzymes PROTEIN SCIENCE Kloeck, C., Khosla, C. 2012; 21 (12): 1781-1791

    Abstract

    Mammalian transglutaminases catalyze post-translational modifications of glutamine residues on proteins and peptides through transamidation or deamidation reactions. Their catalytic mechanism resembles that of cysteine proteases. In virtually every case, their enzymatic activity is modulated by elaborate strategies including controlled gene expression, allostery, covalent modification, and proteolysis. In this review, we focus on our current knowledge of post-translational regulation of transglutaminase activity by physiological as well as synthetic allosteric agents. Our discussion will primarily focus on transglutaminase 2, but will also compare and contrast its regulation with Factor XIIIa as well as transglutaminases 1 and 3. Potential structure-function relationships of known mutations in human transglutaminases are analyzed.

    View details for DOI 10.1002/pro.2162

    View details for Web of Science ID 000311616200001

    View details for PubMedID 23011841

  • Molecular Insights into the Biosynthesis of Guadinomine: A Type III Secretion System Inhibitor JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Holmes, T. C., May, A. E., Zaleta-Riyera, K., Ruby, J. G., Skewes-Cox, P., Fischbach, M. A., DeRisi, J. L., Iwatsuki, M., Omura, S., Khosla, C. 2012; 134 (42): 17797-17806

    Abstract

    Guadinomines are a recently discovered family of anti-infective compounds produced by Streptomyces sp. K01-0509 with a novel mode of action. With an IC(50) of 14 nM, guadinomine B is the most potent known inhibitor of the type III secretion system (TTSS) of Gram-negative bacteria. TTSS activity is required for the virulence of many pathogenic Gram-negative bacteria including Escherichia coli , Salmonella spp., Yersinia spp., Chlamydia spp., Vibrio spp., and Pseudomonas spp. The guadinomine (gdn) biosynthetic gene cluster has been cloned and sequenced and includes 26 open reading frames spanning 51.2 kb. It encodes a chimeric multimodular polyketide synthase, a nonribosomal peptide synthetase, along with enzymes responsible for the biosynthesis of the unusual aminomalonyl-acyl carrier protein extender unit and the signature carbamoylated cyclic guanidine. Its identity was established by targeted disruption of the gene cluster as well as by heterologous expression and analysis of key enzymes in the biosynthetic pathway. Identifying the guadinomine gene cluster provides critical insight into the biosynthesis of these scarce but potentially important natural products.

    View details for DOI 10.1021/ja308622d

    View details for Web of Science ID 000310103800078

    View details for PubMedID 23030602

  • Natural product inhibitors of glucose-6-phosphate translocase MEDCHEMCOMM Charkoudian, L. K., Farrell, B. P., Khosla, C. 2012; 3 (8): 926-931

    View details for DOI 10.1039/c2md20008b

    View details for Web of Science ID 000306759900009

  • Precursor Directed Biosynthesis of an Orthogonally Functional Erythromycin Analogue: Selectivity in the Ribosome Macrolide Binding Pocket JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Harvey, C. J., Puglisi, J. D., Pande, V. S., Cane, D. E., Khosla, C. 2012; 134 (29): 12259-12265

    Abstract

    The macrolide antibiotic erythromycin A and its semisynthetic analogues have been among the most useful antibacterial agents for the treatment of infectious diseases. Using a recently developed chemical genetic strategy for precursor-directed biosynthesis and colony bioassay of 6-deoxyerythromycin D analogues, we identified a new class of alkynyl- and alkenyl-substituted macrolides with activities comparable to that of the natural product. Further analysis revealed a marked and unexpected dependence of antibiotic activity on the size and degree of unsaturation of the precursor. Based on these leads, we also report the precursor-directed biosynthesis of 15-propargyl erythromycin A, a novel antibiotic that not only is as potent as erythromycin A with respect to its ability to inhibit bacterial growth and cell-free ribosomal protein biosynthesis but also harbors an orthogonal functional group that is capable of facile chemical modification.

    View details for DOI 10.1021/ja304682q

    View details for Web of Science ID 000306724500075

    View details for PubMedID 22741553

  • Role of transglutaminase 2 in celiac disease pathogenesis SEMINARS IN IMMUNOPATHOLOGY Klock, C., DiRaimondo, T. R., Khosla, C. 2012; 34 (4): 513-522

    Abstract

    A number of lines of evidence suggest that transglutaminase 2 (TG2) may be one of the earliest disease-relevant proteins to encounter immunotoxic gluten in the celiac gut. These and other investigations also suggest that the reaction catalyzed by TG2 on dietary gluten peptides is essential for the pathogenesis of celiac disease. If so, several questions are of critical significance. How is TG2 activated in the celiac gut? What are the disease-specific and general consequences of activating TG2? Can local inhibition of TG2 in the celiac intestine suppress gluten induced pathogenesis in a dose-responsive manner? And what are the long-term consequences of suppressing TG2 activity in the small intestinal mucosa? Answers to these questions will depend upon the development of judicious models and chemical tools. They also have the potential of yielding powerful next-generation drug candidates for treating this widespread but overlooked chronic disease.

    View details for DOI 10.1007/s00281-012-0305-0

    View details for Web of Science ID 000307305600005

    View details for PubMedID 22437759

  • Role of a Conserved Arginine Residue in Linkers between the Ketosynthase and Acyltransferase Domains of Multimodular Polyketide Synthases BIOCHEMISTRY Yuzawa, S., Kapur, S., Cane, D. E., Khosla, C. 2012; 51 (18): 3708-3710

    Abstract

    The role of interdomain linkers in modular polyketide synthases is poorly understood. Analysis of the 6-deoxyerythronolide B synthase (DEBS) has yielded a model in which chain elongation is governed by interactions between the acyl carrier protein domain and the ketosynthase domain plus an adjacent linker. Alanine scanning mutagenesis of the conserved residues of this linker in DEBS module 3 led to the identification of the R513A mutant with a markedly reduced rate of chain elongation. Limited proteolysis supported a structural role for this Arg. Our findings highlight the importance of domain-linker interactions in assembly line polyketide biosynthesis.

    View details for DOI 10.1021/bi300399u

    View details for Web of Science ID 000303628200002

    View details for PubMedID 22509729

  • Resolving Multiple Protein-Peptide Binding Events: Implication for HLA-DQ2 Mediated Antigen Presentation in Celiac Disease CHEMISTRY-AN ASIAN JOURNAL Wang, J., Jin, X., Liu, J., Khosla, C., Xia, J. 2012; 7 (5): 992-999

    Abstract

    Techniques that can effectively separate protein-peptide complexes from free peptides have shown great value in major histocompatibility complex (MHC)-peptide binding studies. However, most of the available techniques are limited to measuring the binding of a single peptide to an MHC molecule. As antigen presentation in vivo involves both endogenous ligands and exogenous antigens, the deconvolution of multiple binding events necessitates the implementation of a more powerful technique. Here we show that capillary electrophoresis coupled to fluorescence detection (CE-FL) can resolve multiple MHC-peptide binding events owing to its superior resolution and the ability to simultaneously monitor multiple emission channels. We utilized CE-FL to investigate competition and displacement of endogenous peptides by an immunogenic gluten peptide for binding to HLA-DQ2. Remarkably, this immunogenic peptide could displace CLIP peptides from the DQ2 binding site at neutral but not acidic pH. This unusual ability of the gluten peptide supports a direct loading mechanism of antigen presentation in extracellular environment, a property that could explain the antigenicity of dietary gluten in celiac disease.

    View details for DOI 10.1002/asia.201101041

    View details for Web of Science ID 000303239700021

    View details for PubMedID 22411856

  • Combinatorial biosynthesis of polyketides - a perspective CURRENT OPINION IN CHEMICAL BIOLOGY Wong, F. T., Khosla, C. 2012; 16 (1-2): 117-123

    Abstract

    Since their discovery, polyketide synthases have been attractive targets of biosynthetic engineering to make 'unnatural' natural products. Although combinatorial biosynthesis has made encouraging advances over the past two decades, the field remains in its infancy. In this enzyme-centric perspective, we discuss the scientific and technological challenges that could accelerate the adoption of combinatorial biosynthesis as a method of choice for the preparation of encoded libraries of bioactive small molecules. Borrowing a page from the protein structure prediction community, we propose a periodic challenge program to vet the most promising methods in the field, and to foster the collective development of useful tools and algorithms.

    View details for DOI 10.1016/j.cbpa.2012.01.018

    View details for Web of Science ID 000303640400017

    View details for PubMedID 22342766

  • Interferon-gamma Activates Transglutaminase 2 via a Phosphatidylinositol-3-Kinase-Dependent Pathway: Implications for Celiac Sprue Therapy JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS DiRaimondo, T. R., Kloeck, C., Khosla, C. 2012; 341 (1): 104-114

    Abstract

    The mechanism for activation of extracellular transglutaminase 2 (TG2) in the small intestine remains a fundamental mystery in our understanding of celiac sprue pathogenesis. Using the T84 human enterocytic cell line, we show that interferon-γ (IFN-γ), the predominant cytokine secreted by gluten-reactive T cells in the celiac intestine, activates extracellular TG2 in a dose-dependent manner. IFN-γ mediated activation of TG2 requires phosphatidylinositol-3-kinase (PI3K) activity, but is uninfluenced by a number of other kinases reported to be active in T84 cells. Pharmacological inhibition of PI3K in the presence of IFN-γ prevents TG2 activation as well as the previously characterized increase in transepithelial permeability. Our findings therefore establish PI3K as an attractive target for celiac sprue therapy, a possibility that is underscored by the encouraging safety profiles of several PI3K inhibitors undergoing human clinical trials.

    View details for DOI 10.1124/jpet.111.187385

    View details for Web of Science ID 000301530100011

  • Reprogramming a module of the 6-deoxyerythronolide B synthase for iterative chain elongation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kapur, S., Lowry, B., Yuzawa, S., Kenthirapalan, S., Chen, A. Y., Cane, D. E., Khosla, C. 2012; 109 (11): 4110-4115

    Abstract

    Multimodular polyketide synthases (PKSs) have an assembly line architecture in which a set of protein domains, known as a module, participates in one round of polyketide chain elongation and associated chemical modifications, after which the growing chain is translocated to the next PKS module. The ability to rationally reprogram these assembly lines to enable efficient synthesis of new polyketide antibiotics has been a long-standing goal in natural products biosynthesis. We have identified a ratchet mechanism that can explain the observed unidirectional translocation of the growing polyketide chain along the 6-deoxyerythronolide B synthase. As a test of this model, module 3 of the 6-deoxyerythronolide B synthase has been reengineered to catalyze two successive rounds of chain elongation. Our results suggest that high selectivity has been evolutionarily programmed at three types of protein-protein interfaces that are present repetitively along naturally occurring PKS assembly lines.

    View details for DOI 10.1073/pnas.1118734109

    View details for Web of Science ID 000301426700024

    View details for PubMedID 22371562

  • Activation and Inhibition of Transglutaminase 2 in Mice PLOS ONE Dafik, L., Albertelli, M., Stamnaes, J., Sollid, L. M., Khosla, C. 2012; 7 (2)

    Abstract

    Transglutaminase 2 (TG2) is an allosterically regulated enzyme with transamidating, deamidating and cell signaling activities. It is thought to catalyze sequence-specific deamidation of dietary gluten peptides in the small intestines of celiac disease patients. Because this modification has profound consequences for disease pathogenesis, there is considerable interest in the design of small molecule TG2 inhibitors. Although many classes of TG2 inhibitors have been reported, thus far an animal model for screening them to identify promising celiac drug candidates has remained elusive. Using intraperitoneal administration of the toll-like receptor 3 (TLR3) ligand, polyinosinic-polycytidylic acid (poly(I?C)), we induced rapid TG2 activation in the mouse small intestine. Dose dependence was observed in the activation of TG2 as well as the associated villous atrophy, gross clinical response, and rise in serum concentration of the IL-15/IL-15R complex. TG2 activity was most pronounced in the upper small intestine. No evidence of TG2 activation was observed in the lung mucosa, nor were TLR7/8 ligands able to elicit an analogous response. Introduction of ERW1041E, a small molecule TG2 inhibitor, in this mouse model resulted in TG2 inhibition in the small intestine. TG2 inhibition had no effect on villous atrophy, suggesting that activation of this enzyme is a consequence, rather than a cause, of poly(I?C) induced enteropathy. Consistent with this finding, administration of poly(I?C) to TG2 knockout mice also induced villous atrophy. Our findings pave the way for pharmacological evaluation of small molecule TG2 inhibitors as drug candidates for celiac disease.

    View details for DOI 10.1371/journal.pone.0030642

    View details for Web of Science ID 000301979000009

    View details for PubMedID 22319575

  • In vitro and in vivo activity of frenolicin B against Plasmodium falciparum and P berghei JOURNAL OF ANTIBIOTICS Fitzgerald, J. T., Henrich, P. P., O'Brien, C., Krause, M., Ekland, E. H., Mattheis, C., Sa, J. M., Fidock, D., Khosla, C. 2011; 64 (12): 799-801

    View details for DOI 10.1038/ja.2011.94

    View details for Web of Science ID 000298634600008

    View details for PubMedID 22008701

  • Engineered biosynthesis of the antiparasitic agent frenolicin B and rationally designed analogs in a heterologous host JOURNAL OF ANTIBIOTICS Fitzgerald, J. T., Ridley, C. P., Khosla, C. 2011; 64 (12): 759-762

    Abstract

    The polyketide antibiotic frenolicin B harbors a biosynthetically intriguing benzoisochromanequinone core, and has been shown to exhibit promising antiparasitic activity against Eimeria tenella. To facilitate further exploration of its chemistry and biology, we constructed a biosynthetic route to frenolicin B in the heterologous host Streptomyces coelicolor CH999, despite the absence of key enzymes in the identified frenolicin gene cluster. Together with our understanding of the underlying polyketide biosynthetic pathway, this heterologous production system was exploited to produce analogs modified at the C15 position. Both the natural product and these analogs inhibited the growth of Toxoplasma gondii in a manner that reveals sensitivity to the length of the C15 substituent. The ability to construct a functional biosynthetic pathway, despite a lack of genetic information, illustrates the feasibility of a modular approach to engineering medicinally relevant polyketide products.

    View details for DOI 10.1038/ja.2011.86

    View details for Web of Science ID 000298634600001

    View details for PubMedID 21934692

  • In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yu, X., Liu, T., Zhu, F., Khosla, C. 2011; 108 (46): 18643-18648

    Abstract

    Microbial fatty acid derivatives are emerging as promising alternatives to fossil fuel derived transportation fuels. Among bacterial fatty acid synthases (FAS), the Escherichia coli FAS is perhaps the most well studied, but little is known about its steady-state kinetic behavior. Here we describe the reconstitution of E. coli FAS using purified protein components and report detailed kinetic analysis of this reconstituted system. When all ketosynthases are present at 1 ?M, the maximum rate of free fatty acid synthesis of the FAS exceeded 100 ?M/ min. The steady-state turnover frequency was not significantly inhibited at high concentrations of any substrate or cofactor. FAS activity was saturated with respect to most individual protein components when their concentrations exceeded 1 ?M. The exceptions were FabI and FabZ, which increased FAS activity up to concentrations of 10 ?M; FabH and FabF, which decreased FAS activity at concentrations higher than 1 ?M; and holo-ACP and TesA, which gave maximum FAS activity at 30 ?M concentrations. Analysis of the S36T mutant of the ACP revealed that the unusual dependence of FAS activity on holo-ACP concentration was due, at least in part, to the acyl-phosphopantetheine moiety. MALDI-TOF mass spectrometry analysis of the reaction mixture further revealed medium and long chain fatty acyl-ACP intermediates as predominant ACP species. We speculate that one or more of such intermediates are key allosteric regulators of FAS turnover. Our findings provide a new basis for assessing the scope and limitations of using E. coli as a biocatalyst for the production of diesel-like fuels.

    View details for DOI 10.1073/pnas.1110852108

    View details for Web of Science ID 000297008900026

    View details for PubMedID 22042840

  • Activation of Extracellular Transglutaminase 2 by Thioredoxin JOURNAL OF BIOLOGICAL CHEMISTRY Jin, X., Stamnaes, J., Klock, C., DiRaimondo, T. R., Sollid, L. M., Khosla, C. 2011; 286 (43): 37866-37873

    Abstract

    The mechanism of activation of transglutaminase 2 (TG2) in the extracellular matrix remains a fundamental mystery in our understanding of the biology of this multifunctional mammalian enzyme. Earlier investigations have highlighted the role of a disulfide bond formed by vicinal Cys residues in maintaining calcium-bound TG2 in an inactive state. Here, we have shown that the redox potential of this disulfide bond is approximately -190 mV, a high value for a disulfide bond in proteins. Consistent with this observation, TG2 activity in a freshly wounded fibroblast culture depends upon the redox potential of the environment. We sought to identify a physiological mechanism for the activation of oxidized TG2. With a k(cat)/K(m) of 1.6 ?m(-1) min(-1), human thioredoxin (Trx) was a highly specific activator of oxidized human TG2. Trx-mediated activation of TG2 was blocked by PX-12, a small molecule Trx inhibitor that is undergoing clinical trials as a cancer chemotherapeutic agent. In a mixed culture containing fibroblasts and monocytic cells, interferon-? stimulated Trx release from monocytes, which in turn activated TG2 around the fibroblasts. Recombinant human Trx could also activate extracellular TG2 in cryosections of human and mouse small intestinal biopsies. In addition to explaining how TG2 can be activated by dietary gluten in the small intestinal mucosa of celiac sprue patients, our findings reveal a new strategy for inhibiting the undesirable consequences of TG2 activity in this widespread, lifelong disease.

    View details for DOI 10.1074/jbc.M111.287490

    View details for Web of Science ID 000296542400081

    View details for PubMedID 21908620

  • Structural and Biochemical Studies of the Hedamycin Type II Polyketide Ketoreductase (HedKR): Molecular Basis of Stereo- and Regiospecificities BIOCHEMISTRY Javidpour, P., Das, A., Khosla, C., Shiou-Chuan Tsai, S. C. 2011; 50 (34): 7426-7439

    Abstract

    Bacterial aromatic polyketides that include many antibiotic and antitumor therapeutics are biosynthesized by the type II polyketide synthase (PKS), which consists of 5-10 stand-alone enzymatic domains. Hedamycin, an antitumor antibiotic polyketide, is uniquely primed with a hexadienyl group generated by a type I PKS followed by coupling to a downstream type II PKS to biosynthesize a 24-carbon polyketide, whose C9 position is reduced by hedamycin type II ketoreductase (hedKR). HedKR is homologous to the actinorhodin KR (actKR), for which we have conducted extensive structural studies previously. How hedKR can accommodate a longer polyketide substrate than the actKR, and the molecular basis of its regio- and stereospecificities, is not well understood. Here we present a detailed study of hedKR that sheds light on its specificity. Sequence alignment of KRs predicts that hedKR is less active than actKR, with significant differences in substrate/inhibitor recognition. In vitro and in vivo assays of hedKR confirmed this hypothesis. The hedKR crystal structure further provides the molecular basis for the observed differences between hedKR and actKR in the recognition of substrates and inhibitors. Instead of the 94-PGG-96 motif observed in actKR, hedKR has the 92-NGG-94 motif, leading to S-dominant stereospecificity, whose molecular basis can be explained by the crystal structure. Together with mutations, assay results, docking simulations, and the hedKR crystal structure, a model for the observed regio- and stereospecificities is presented herein that elucidates how different type II KRs recognize substrates with different chain lengths, yet precisely reduce only the C9-carbonyl group. The molecular features of hedKR important for regio- and stereospecificities can potentially be applied to biosynthesize new polyketides via protein engineering that rationally controls polyketide ketoreduction.

    View details for DOI 10.1021/bi2006866

    View details for Web of Science ID 000294083600012

    View details for PubMedID 21776967

  • Analysis of the Ketosynthase-Chain Length Factor Heterodimer from the Fredericamycin Polyketide Synthase CHEMISTRY & BIOLOGY Szu, P., Govindarajan, S., Meehan, M. J., Das, A., Don D Nguyen, D. D., Dorrestein, P. C., Minshull, J., Khosla, C. 2011; 18 (8): 1021-1031

    Abstract

    The pentadecaketide fredericamycin has the longest carbon chain backbone among polycyclic aromatic polyketide antibiotics whose biosynthetic genes have been sequenced. This backbone is synthesized by the bimodular fdm polyketide synthase (PKS). Here, we demonstrate that the bimodular fdm PKS as well as its elongation module alone synthesize undecaketides and dodecaketides. Thus, unlike other homologs, the fdm ketosynthase-chain length factor (KS-CLF) heterodimer does not exclusively control the backbone length of its natural product. Using sequence- and structure-based approaches, 48 CLF multiple mutants were engineered and analyzed. Unexpectedly, the I134F mutant was unable to turn over but could initiate and partially elongate the polyketide chain. This unprecedented mutant suggests that the KS-CLF heterodimer harbors an as yet uncharacterized chain termination mechanism. Together, our findings reveal fundamental mechanistic differences between the fdm PKS and its well-studied homologs.

    View details for DOI 10.1016/j.chembiol.2011.07.015

    View details for Web of Science ID 000295203200012

    View details for PubMedID 21867917

  • Structure and Mechanism of the trans-Acting Acyltransferase from the Disorazole Synthase BIOCHEMISTRY Wong, F. T., Jin, X., Mathews, I. I., Cane, D. E., Khosla, C. 2011; 50 (30): 6539-6548

    Abstract

    The 1.51 Å resolution X-ray crystal structure of the trans-acyltransferase (AT) from the "AT-less" disorazole synthase (DSZS) and that of its acetate complex at 1.35 Å resolution are reported. Separately, comprehensive alanine-scanning mutagenesis of one of its acyl carrier protein substrates (ACP1 from DSZS) led to the identification of a conserved Asp45 residue on the ACP, which contributes to the substrate specificity of this unusual enzyme. Together, these experimental findings were used to derive a model for the selective association of the DSZS AT and its ACP substrate. With a goal of structurally characterizing the AT-ACP interface, a strategy was developed for covalently cross-linking the active site Ser ? Cys mutant of the DSZS AT to its ACP substrate and for purifying the resulting AT-ACP complex to homogeneity. The S86C DSZS AT mutant was found to be functional, albeit with a transacylation efficiency 200-fold lower than that of its wild-type counterpart. Our findings provide new insights as well as new opportunities for high-resolution analysis of an important protein-protein interface in polyketide synthases.

    View details for DOI 10.1021/bi200632j

    View details for Web of Science ID 000293035500003

    View details for PubMedID 21707057

  • Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase PROTEIN SCIENCE Charkoudian, L. K., Liu, C. W., Capone, S., Kapur, S., Cane, D. E., Togni, A., Seebach, D., Khosla, C. 2011; 20 (7): 1244-1255

    Abstract

    The assembly-line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6-deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein-protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF?-S-ACP, was developed as a ¹?F NMR spectroscopic probe of protein-protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.

    View details for DOI 10.1002/pro.652

    View details for Web of Science ID 000292257600017

    View details for PubMedID 21563224

  • Novel chemo-sensitizing agent, ERW1227B, impairs cellular motility and enhances cell death in glioblastomas JOURNAL OF NEURO-ONCOLOGY Yuan, L., Holmes, T. C., Watts, R. E., Khosla, C., Broekelmann, T. J., Mecham, R., Zheng, H., Izaguirre, E. W., Rich, K. M. 2011; 103 (2): 207-219

    Abstract

    Glioblastomas display variable phenotypes that include increased drug-resistance associated with enhanced migratory and anti-apoptotic characteristics. These shared characteristics contribute to failure of clinical treatment regimens. Identification of novel compounds that promote cell death and impair cellular motility is a logical strategy to develop more effective clinical protocols. We recently described the ability of the small molecule, KCC009, a tissue transglutaminase (TG2) inhibitor, to sensitize glioblastoma cells to chemotherapy. In the current study, we synthesized a series of related compounds that show variable ability to promote cell death and impair motility in glioblastomas, irrespective of their ability to inhibit TG2. Each compound has a 3-bromo-4,5-dihydroisoxazole component that presumably reacts with nucleophilic cysteine thiol residues in the active sites of proteins that have an affinity to the small molecule. Our studies focused on the effects of the compound, ERW1227B. Treatment of glioblastoma cells with ERW1227B was associated with both down-regulation of the PI-3 kinase/Akt pathway, which enhanced cell death; as well as disruption of focal adhesive complexes and intracellular actin fibers, which impaired cellular mobility. Bioassays as well as time-lapse photography of glioblastoma cells treated with ERW1227B showed cell death and rapid loss of cellular motility. Mice studies with in vivo glioblastoma models demonstrated the ability of ERW1227B to sensitize tumor cells to cell death after treatment with either chemotherapy or radiation. The above findings identify ERW1227B as a potential novel therapeutic agent in the treatment of glioblastomas.

    View details for DOI 10.1007/s11060-010-0379-2

    View details for Web of Science ID 000290773100004

    View details for PubMedID 20824305

  • Novel therapies for coeliac disease JOURNAL OF INTERNAL MEDICINE Sollid, L. M., Khosla, C. 2011; 269 (6): 604-613

    Abstract

    Coeliac disease is a widespread, lifelong disorder for which dietary control represents the only accepted form of therapy. There is an unmet need for nondietary therapies to treat this condition. Most ongoing and emerging drug-discovery programmes are based on the understanding that coeliac disease is caused by an inappropriate T-cell-mediated immune response to dietary gluten proteins. Recent genome-wide association studies lend further support to this pathogenic model. The central role of human leucocyte antigen genes has been validated, and a number of new risk loci have been identified, most of which are related to the biology of T cells and antigen-presenting cells. Here, we review the status of potential nondietary therapies under consideration for coeliac disease. We conclude that future development of novel therapies will be aided considerably by the identification of new, preferably noninvasive, surrogate markers for coeliac disease activity.

    View details for DOI 10.1111/j.1365-2796.2011.02376.x

    View details for Web of Science ID 000290867900006

    View details for PubMedID 21401739

  • Acylideneoxoindoles: a new class of reversible inhibitors of human transglutaminase 2. Bioorganic & medicinal chemistry letters Klöck, C., Jin, X., Choi, K., Khosla, C., Madrid, P. B., Spencer, A., Raimundo, B. C., Boardman, P., Lanza, G., Griffin, J. H. 2011; 21 (9): 2692-2696

    Abstract

    Inhibitors of human transglutaminase 2 (TG2) are anticipated to be useful in the therapy of a variety of diseases including celiac sprue as well as certain CNS disorders and cancers. A class of 3-acylidene-2-oxoindoles was identified as potent reversible inhibitors of human TG2. Structure-activity relationship analysis of a lead compound led to the generation of several potent, competitive inhibitors. Analogs with significant non-competitive character were also identified, suggesting that the compounds bind at one or more allosteric regulatory sites on this multidomain enzyme. The most active compounds had K(i) values below 1.0 μM in two different kinetic assays for human TG2, and may therefore be suitable for investigations into the role of TG2 in physiology and disease in animals.

    View details for DOI 10.1016/j.bmcl.2010.12.037

    View details for PubMedID 21215619

  • Chemistry and Biology of Macrolide Antiparasitic Agents JOURNAL OF MEDICINAL CHEMISTRY Lee, Y., Choi, J. Y., Fu, H., Harvey, C., Ravindran, S., Roush, W. R., Boothroyd, J. C., Khosla, C. 2011; 54 (8): 2792-2804

    Abstract

    Macrolide antibacterial agents inhibit parasite proliferation by targeting the apicoplast ribosome. Motivated by the long-term goal of identifying antiparasitic macrolides that lack antibacterial activity, we have systematically analyzed the structure-activity relationships among erythromycin analogues and have also investigated the mechanism of action of selected compounds. Two lead compounds, N-benzylazithromycin (11) and N-phenylpropylazithromycin (30), were identified with significantly higher antiparasitic activity and lower antibacterial activity than erythromycin or azithromycin. Molecular modeling based on the cocrystal structure of azithromycin bound to the bacterial ribosome suggested that a substituent at the N-9 position of desmethylazithromycin could improve selectivity because of species-specific interactions with the ribosomal L22 protein. Like other macrolides, these lead compounds display a strong "delayed death phenotype"; however, their early effects on T. gondii replication are more pronounced.

    View details for DOI 10.1021/jm101593u

    View details for Web of Science ID 000289697800018

    View details for PubMedID 21428405

  • Dihydroisoxazole Analogs for Labeling and Visualization of Catalytically Active Transglutaminase 2 CHEMISTRY & BIOLOGY Dafik, L., Khosla, C. 2011; 18 (1): 58-66

    Abstract

    We report the synthesis and preliminary characterization of "clickable" inhibitors of human transglutaminase 2 (TG2). These inhibitors possess the 3-halo-4,5-dihydroisoxazole warhead along with bioorthogonal groups such as azide or alkyne moieties that enable subsequent covalent modification with fluorophores. Their mechanism for inhibition of TG2 is based on halide displacement, resulting in the formation of a stable imino thioether. Inhibition assays against recombinant human TG2 revealed that some of the clickable inhibitors prepared in this study have comparable specificity as benchmark dihydroisoxazole inhibitors reported earlier. At low micromolar concentrations they completely inhibited transiently activated TG2 in a WI-38 fibroblast scratch assay and could subsequently be used to visualize the active enzyme in situ. The potential use of these inhibitors to probe the role of TG2 in celiac sprue as well as other diseases is discussed.

    View details for DOI 10.1016/j.chembiol.2010.11.004

    View details for Web of Science ID 000287540300011

    View details for PubMedID 21276939

  • Improved precursor-directed biosynthesis in E. coli via directed evolution JOURNAL OF ANTIBIOTICS Lee, H. Y., Harvey, C. J., Cane, D. E., Khosla, C. 2011; 64 (1): 59-64

    Abstract

    Erythromycin and related macrolide antibiotics are widely used polyketide natural products. We have evolved an engineered biosynthetic pathway in Escherichia coli that yields erythromycin analogs from simple synthetic precursors. Multiple rounds of mutagenesis and screening led to the identification of new mutant strains with improved efficiency for precursor-directed biosynthesis. Genetic and biochemical analysis suggested that the phenotypically relevant alterations in these mutant strains were localized exclusively to the host-vector system, and not to the polyketide synthase. We also demonstrate the utility of this improved system through engineered biosynthesis of a novel alkynyl erythromycin derivative with comparable antibacterial activity to its natural counterpart. In addition to reinforcing the power of directed evolution for engineering macrolide biosynthesis, our studies have identified a new lead substance for investigating structure-function relationships in the bacterial ribosome.

    View details for DOI 10.1038/ja.2010.129

    View details for Web of Science ID 000287072300009

    View details for PubMedID 21081955

  • Molecular recognition between ketosynthase and acyl carrier protein domains of the 6-deoxyerythronolide B synthase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kapur, S., Chen, A. Y., Cane, D. E., Khosla, C. 2010; 107 (51): 22066-22071

    Abstract

    Every polyketide synthase module has an acyl carrier protein (ACP) and a ketosynthase (KS) domain that collaborate to catalyze chain elongation. The same ACP then engages the KS domain of the next module to facilitate chain transfer. Understanding the mechanism for this orderly progress of the growing polyketide chain represents a fundamental challenge in assembly line enzymology. Using both experimental and computational approaches, the molecular basis for KS-ACP interactions in the 6-deoxyerythronolide B synthase has been decoded. Surprisingly, KS-ACP recognition is controlled at different interfaces during chain elongation versus chain transfer. In fact, chain elongation is controlled at a docking site remote from the catalytic center. Not only do our findings reveal a new principle in the modular control of polyketide antibiotic biosynthesis, they also provide a rationale for the mandatory homodimeric structure of polyketide synthases, in contrast to the monomeric nonribosomal peptide synthetases.

    View details for DOI 10.1073/pnas.1014081107

    View details for Web of Science ID 000285521800027

    View details for PubMedID 21127271

  • Stereospecificity of the Dehydratase Domain of the Erythromycin Polyketide Synthase JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Valenzano, C. R., You, Y., Garg, A., Keatinge-Clay, A., Khosla, C., Cane, D. E. 2010; 132 (42): 14697-14699

    Abstract

    The dehydratase (DH) domain of module 4 of the 6-deoxyerythronolide B synthase (DEBS) has been shown to catalyze an exclusive syn elimination/syn addition of water. Incubation of recombinant DH4 with chemoenzymatically prepared anti-(2R,3R)-2-methyl-3-hydroxypentanoyl-ACP (2a-ACP) gave the dehydration product 3-ACP. Similarly, incubation of DH4 with synthetic 3-ACP resulted in the reverse enzyme-catalyzed hydration reaction, giving an ?3:1 equilbrium mixture of 2a-ACP and 3-ACP. Incubation of a mixture of propionyl-SNAC (4), methylmalonyl-CoA, and NADPH with the DEBS ?-ketoacyl synthase-acyl transferase [KS6][AT6] didomain, DEBS ACP6, and the ketoreductase domain from tylactone synthase module 1 (TYLS KR1) generated in situ anti-2a-ACP that underwent DH4-catalyzed syn dehydration to give 3-ACP. DH4 did not dehydrate syn-(2S,3R)-2b-ACP, syn-(2R,3S)-2c-ACP, or anti-(2S,3S)-2d-ACP generated in situ by DEBS KR1, DEBS KR6, or the rifamycin synthase KR7 (RIFS KR7), respectively. Similarly, incubation of a mixture of (2S,3R)-2-methyl-3-hydroxypentanoyl-N-acetylcysteamine thioester (2b-SNAC), methylmalonyl-CoA, and NADPH with DEBS [KS6][AT6], DEBS ACP6, and TYLS KR1 gave anti-(2R,3R)-6-ACP that underwent syn dehydration catalyzed by DEBS DH4 to give (4R,5R)-(E)-2,4-dimethyl-5-hydroxy-hept-2-enoyl-ACP (7-ACP). The structure and stereochemistry of 7 were established by GC-MS and LC-MS comparison of the derived methyl ester 7-Me to a synthetic sample of 7-Me.

    View details for DOI 10.1021/ja107344h

    View details for Web of Science ID 000283403200003

    View details for PubMedID 20925342

  • A Balancing Act for Taxol Precursor Pathways in E. coli SCIENCE Liu, T., Khosla, C. 2010; 330 (6000): 44-45

    View details for DOI 10.1126/science.1195014

    View details for Web of Science ID 000282334500025

    View details for PubMedID 20929799

  • In Living Color: Bacterial Pigments as an Untapped Resource in the Classroom and Beyond PLOS BIOLOGY Charkoudian, L. K., Fitzgerald, J. T., Khosla, C., Champlin, A. 2010; 8 (10)

    View details for DOI 10.1371/journal.pbio.1000510

    View details for Web of Science ID 000283495100011

    View details for PubMedID 20957190

  • Thematic Minireview Series on Antibacterial Natural Products: New Tricks for Old Dogs JOURNAL OF BIOLOGICAL CHEMISTRY Khosla, C. 2010; 285 (36): 27499-27499

    View details for DOI 10.1074/jbc.R110.150417

    View details for Web of Science ID 000281404100002

    View details for PubMedID 20522550

  • Redox Regulation of Transglutaminase 2 Activity JOURNAL OF BIOLOGICAL CHEMISTRY Stamnaes, J., Pinkas, D. M., Fleckenstein, B., Khosla, C., Sollid, L. M. 2010; 285 (33): 25402-25409

    Abstract

    Transglutaminase 2 (TG2) in the extracellular matrix is largely inactive but is transiently activated upon certain types of inflammation and cell injury. The enzymatic activity of extracellular TG2 thus appears to be tightly regulated. As TG2 is known to be sensitive to changes in the redox environment, inactivation through oxidation presents a plausible mechanism. Using mass spectrometry, we have identified a redox-sensitive cysteine triad consisting of Cys(230), Cys(370), and Cys(371) that is involved in oxidative inactivation of TG2. Within this triad, Cys(370) was found to participate in disulfide bonds with both Cys(230) and its neighbor, Cys(371). Notably, Ca(2+) was found to protect against formation of these disulfide bonds. To investigate the role of each cysteine residue, we created alanine mutants and found that Cys(230) appears to promote oxidation and inactivation of TG2 by facilitating formation of Cys(370)-Cys(371) through formation of the Cys(230)-Cys(370) disulfide bond. Although vicinal disulfide pairs are found in several transglutaminase isoforms, Cys(230) is unique for TG2, suggesting that this residue acts as an isoform-specific redox sensor. Our findings suggest that oxidation is likely to influence the amount of active TG2 present in the extracellular environment.

    View details for DOI 10.1074/jbc.M109.097162

    View details for Web of Science ID 000280682400033

    View details for PubMedID 20547769

  • Mechanism and Engineering of Polyketide Chain Initiation in Fredericamycin Biosynthesis JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Das, A., Szu, P., Fitzgerald, J. T., Khosla, C. 2010; 132 (26): 8831-?

    Abstract

    The ability to incorporate atypical primer units through the use of dedicated initiation polyketide synthase (PKS) modules offers opportunities to expand the molecular diversity of polyketide natural products. Here we identify the initiation PKS module responsible for hexadienyl priming of the antibiotic fredericamycin and investigate its biochemical properties. We also exploit this PKS module for the design and in vivo biosynthesis of unusually primed analogues of a representative polyketide product, thereby emphasizing its utility to the metabolic engineer.

    View details for DOI 10.1021/ja102517q

    View details for Web of Science ID 000279561200009

    View details for PubMedID 20540492

  • Cloning, Sequencing, Heterologous Expression, and Mechanistic Analysis of A-74528 Biosynthesis JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Zaleta-Rivera, K., Charkoudian, L. K., Ridley, C. P., Khosla, C. 2010; 132 (26): 9122-9128

    Abstract

    A-74528 is a recently discovered natural product of Streptomyces sp. SANK 61196 that inhibits 2',5'-oligoadenylate phosphodiesterase (2'-PDE), a key regulatory enzyme of the interferon pathway. Inhibition of 2'-PDE by A-74528 reduces viral replication, and therefore shows promise as a new type of antiviral drug. The complete A-74528 gene cluster, comprising 29 open reading frames, was cloned and sequenced, and shown to possess a type II polyketide synthase (PKS) at its core. Its identity was confirmed by analysis of a mutant generated by targeted disruption of a PKS gene, and by functional expression in a heterologous Streptomyces host. Remarkably, it showed exceptional end-to-end sequence identity to the gene cluster responsible for biosynthesis of fredericamycin A, a structurally unrelated antitumor antibiotic with a distinct mode of action. Whereas the fredericamycin producing strain, Streptomyces griseus, produced undetectable quantities of A-74528, the A-74528 gene cluster was capable of producing both antibiotics. The biosynthetic roles of three genes, including one that represents the only qualitative difference between the two gene clusters, were investigated by targeted gene disruption. The implications for the evolution of antibiotics with different biological activities from the same gene cluster are discussed.

    View details for DOI 10.1021/ja102519v

    View details for Web of Science ID 000279561200070

    View details for PubMedID 20550125

  • Quantitative analysis and engineering of fatty acid biosynthesis in E. coli METABOLIC ENGINEERING Liu, T., Vora, H., Khosla, C. 2010; 12 (4): 378-386

    Abstract

    Fatty acids are central hydrocarbon intermediates in the biosynthesis of diesel from renewable sources. We have engineered an Escherichia coli cell line that produces 4.5 g/L/day total fatty acid in a fed-batch fermentation. However, further enhancement of fatty acid biosynthesis in this cell line proved unpredictable. To develop a more reliable engineering strategy, a cell-free system was developed that enabled direct, quantitative investigation of fatty acid biosynthesis and its regulation in E. coli. Using this system, the strong dependence of fatty acid synthesis on malonyl-CoA availability and several important phenomena in fatty acid synthesis were verified. Results from this cell-free system were confirmed via the generation and analysis of metabolically engineered strains of E. coli. Our quantitative findings highlight the enormous catalytic potential of the E. coli fatty acid biosynthetic pathway, and target specific steps for protein and metabolic engineering to enhance the catalytic conversion of glucose into biodiesel.

    View details for DOI 10.1016/j.ymben.2010.02.003

    View details for Web of Science ID 000278547700007

    View details for PubMedID 20184964

  • Characterization of transglutaminase type II role in dendritic cell differentiation and function JOURNAL OF LEUKOCYTE BIOLOGY Matic, I., Sacchi, A., Rinaldi, A., Melino, G., Khosla, C., Falasca, L., Piacentini, M. 2010; 88 (1): 181-188

    Abstract

    DCs play an essential role in the endotoxic shock, and their profound depletion occurs in septic patients and septic mice. TG2(-/-) mice are more resistant to the endotoxic shock induced by LPS. Here, we studied the cellular and molecular basis of this effect, analyzing the role of the enzyme in DC maturation and function. We show that TG2 is up-regulated drastically during the final, functional maturation of DCs consequent to LPS treatment. In keeping with this finding, the inhibition of the enzyme cross-linking activity determines the impairment of DC function highlighted by wide phenotypic changes associated with a reduced production of cytokines (IL-10, IL-12) after LPS treatment and a lower ability to induce IFN-gamma production by naïve T cells. The in vivo analysis of DCs obtained from TG2(-/-) mice confirmed that the enzyme ablation leads to an impairment of DC maturation and their reduced responsiveness to LPS treatment. In fact, a marked decrease in DC death, TLR4 down-regulation, and impaired up-regulation of MHCII and CD86 were observed in TG2(-/-) mice. Taken together, these data suggest that TG2 plays an important role in regulating the response of DCs to LPS and could be a candidate target for treating endotoxin-induced sepsis.

    View details for DOI 10.1189/jlb.1009691

    View details for Web of Science ID 000279356700019

    View details for PubMedID 20371597

  • Novel aspects of quantitation of immunogenic wheat gluten peptides by liquid chromatography-mass spectrometry/mass spectrometry JOURNAL OF CHROMATOGRAPHY A Sealey-Voyksner, J. A., Khosla, C., Voyksner, R. D., Jorgenson, J. W. 2010; 1217 (25): 4167-4183

    Abstract

    A novel, specific and sensitive non-immunological liquid chromatography-mass spectrometry (LC-MS) based assay has been developed to detect and quantify trace levels of wheat gluten in food and consumer products. Detection and quantification of dietary gluten is important, because gluten is a principle trigger of a variety of immune diseases including food allergies and intolerances. One such disease, celiac sprue, can cause intestinal inflammation and enteropathy in patients who are exposed to dietary gluten. At present, immunochemistry is the leading analytical method for gluten detection in food. Consequently, enzyme-linked immunosorbent assays (ELISAs), such as the sandwich or competitive type assays, are the only commercially available methods to ensure that food and consumer products are accurately labeled as gluten-free. The availability of a comprehensive, fast and economic alternative to the immunological ELISA may also facilitate research towards the development of new drugs, therapies and food processing technologies to aid patients with gluten intolerances and for gluten-free labeling and certification purposes. LC-MS is an effective and efficient analytical technique for the study of cereal grain proteins and to quantify trace levels of targeted dietary gluten peptides in complex matrices. Initial efforts in this area afforded the unambiguous identification and structural characterization of six unique physiologically relevant wheat gluten peptides. This paper describes the development and optimization of an LC-MS/MS method that attempts to provide the best possible accuracy and sensitivity for the quantitative detection of trace levels of these six peptides in various food and consumer products. The overall performance of this method was evaluated using native cereal grains. Experimental results demonstrated that this method is capable of detecting and quantifying select target peptides in food over a range from 10pg/mg to 100ng/mg (corresponding to approximately 0.01-100ppm). Limits of detection (LOD) and quantification (LOQ) for the six target peptides were determined to range from 1 to 30pg/mg and 10-100pg/mg respectively. Reproducibility of the assay was demonstrated by evaluation of calibration data as well as data collected from the analysis of quality control standards over a period of four consecutive days. The average coefficient of determination (R(2)) for each peptide was consistently found to be >0.995 with residuals ranging from approximately 80% to 110%. Spike recovery data for each peptide in various matrices was evaluated at a concentration level near the approximate LOQ for each, as well as at higher concentration levels (30 and 60ng/mg). The average range of accuracy of detection for all peptides at the lower concentration level was determined to be 90% (+/-11), while accuracy at the 30 and 60ng/mg levels was 98% (+/-5%) and 98% (+/-3%), respectively. The usefulness and capabilities of this method are presented in a practical application to prospectively screen a variety of common commercially available (native and processed) gluten-containing and gluten-free foods and products.

    View details for DOI 10.1016/j.chroma.2010.01.067

    View details for Web of Science ID 000278779000026

    View details for PubMedID 20181349

  • Inhibition of Tubulogenesis and of Carcinogen-mediated Signaling in Brain Endothelial Cells Highlight the Antiangiogenic Properties of a Mumbaistatin Analog CHEMICAL BIOLOGY & DRUG DESIGN Tahanian, E., Lord-Dufour, S., Das, A., Khosla, C., Roy, R., Annabi, B. 2010; 75 (5): 481-488

    Abstract

    A better understanding of the metabolic adaptations of the vascular endothelial cells (EC) that mediate tumor vascularization would help the development of new drugs and therapies. Novel roles in cell survival and metabolic adaptation to hypoxia have been ascribed to the microsomal glucose-6-phosphate translocase (G6PT). While antitumorigenic properties of G6PT inhibitors such as chlorogenic acid (CHL) have been documented, those of the G6PT inhibitor and semi-synthetic analog AD4-015 of the polyketide mumbaistatin are not understood. In the present study, we evaluated the in vitro antiangiogenic impact of AD4-015 on human brain microvascular endothelial cells (HBMEC), which play an essential role as structural and functional components in tumor angiogenesis. We found that in vitro HBMEC migration and tubulogenesis were reduced by AD4-015 but not by CHL. The mumbaistatin analog significantly inhibited the phorbol 12-myristate 13-acetate (PMA)-induced matrix-metalloproteinase (MMP)-9 secretion and gene expression as assessed by zymography and RT-PCR. PMA-mediated cell signaling leading to cyclooxygenase (COX)-2 expression and IkappaB downregulation was also inhibited, further confirming AD4-015 as a cell signaling inhibitor in tumor promoting conditions. G6PT functions may therefore account for the metabolic flexibility that enables EC-mediated neovascularization. This process could be specifically targeted within the vasculature of developing brain tumors by G6PT inhibitors.

    View details for DOI 10.1111/j.1747-0285.2010.00961.x

    View details for Web of Science ID 000275949500006

    View details for PubMedID 20486934

  • Visualization of Transepithelial Passage of the Immunogenic 33-Residue Peptide from alpha-2 Gliadin in Gluten-Sensitive Macaques PLOS ONE Mazumdar, K., Alvarez, X., Borda, J. T., Dufour, J., Martin, E., Bethune, M. T., Khosla, C., Sestak, K. 2010; 5 (4)

    Abstract

    Based on clinical, histopathological and serological similarities to human celiac disease (CD), we recently established the rhesus macaque model of gluten sensitivity. In this study, we further characterized this condition based on presence of anti-tissue transglutaminase 2 (TG2) antibodies, increased intestinal permeability and transepithelial transport of a proteolytically resistant, immunotoxic, 33-residue peptide from alpha(2)-gliadin in the distal duodenum of gluten-sensitive macaques.Six rhesus macaques were selected for study from a pool of 500, including two healthy controls and four gluten-sensitive animals with elevated anti-gliadin or anti-TG2 antibodies as well as history of non-infectious chronic diarrhea. Pediatric endoscope-guided pinch biopsies were collected from each animal's distal duodenum following administration of a gluten-containing diet (GD) and again after remission by gluten-free diet (GFD). Control biopsies always showed normal villous architecture, whereas gluten-sensitive animals on GD exhibited histopathology ranging from mild lymphocytic infiltration to villous atrophy, typical of human CD. Immunofluorescent microscopic analysis of biopsies revealed IgG+ and IgA+ plasma-like cells producing antibodies that colocalized with TG2 in gluten-sensitive macaques only. Following instillation in vivo, the Cy-3-labeled 33-residue gluten peptide colocalized with the brush border protein villin in all animals. In a substantially enteropathic macaque with "leaky" duodenum, the peptide penetrated beneath the epithelium into the lamina propria.The rhesus macaque model of gluten sensitivity not only resembles the histopathology of CD but it also may provide a model for studying intestinal permeability in states of epithelial integrity and disrepair.

    View details for DOI 10.1371/journal.pone.0010228

    View details for Web of Science ID 000276853800018

    View details for PubMedID 20419103

  • Protein-Protein Recognition between Acyltransferases and Acyl Carrier Proteins in Multimodular Polyketide Synthases BIOCHEMISTRY Wong, F. T., Chen, A. Y., Cane, D. E., Khosla, C. 2010; 49 (1): 95-102

    Abstract

    Acyltransferase (AT) domains of multimodular polyketide synthases are the primary gatekeepers for stepwise incorporation of building blocks into a growing polyketide chain. Each AT domain has two substrates, an alpha-carboxylated CoA thioester (e.g., malonyl-CoA or methylmalonyl-CoA) and an acyl carrier protein (ACP). Whereas the acyl-CoA specificity of AT domains has been extensively investigated, little is known about their ACP specificity. Guided by recent high-resolution structural insights, we have systematically probed the protein-protein interactions between AT domains, ACP domains, and the linkers that flank AT domains. Representative AT domains of the 6-deoxyerythronolide B synthase (DEBS) have greater than 10-fold specificity for their cognate ACP substrates as compared to other ACP domains from the same synthase. Both of the flanking (N- and C-terminal) linkers of an AT domain contributed to the efficiency and specificity of transacylation. As a frame of reference, the activity and specificity of a stand-alone AT domain from the "AT-less" disorazole synthase (DSZS) were also quantified. The activity (k(cat)/K(M)) of this AT was >250-fold higher than the corresponding values for DEBS AT domains. Although the AT from DSZS discriminated modestly against ACP domains from DEBS, it exhibited >40-fold higher activity in trans in the presence of these heterologous substrates than their natural AT domains. Our results highlight the opportunity for regioselective modification of a polyketide backbone by in trans complementation of inactivated AT domains. They also reinforce the need for more careful consideration of protein-protein interactions in the engineering of these assembly line enzymes.

    View details for DOI 10.1021/bi901826g

    View details for Web of Science ID 000273267300012

    View details for PubMedID 19921859

  • Genetic Engineering of Escherichia coli for Biofuel Production ANNUAL REVIEW OF GENETICS, VOL 44 Liu, T., Khosla, C. 2010; 44: 53-69

    Abstract

    In order to mitigate climate change without adversely affecting global energy supply, there is growing interest in the possibility of producing transportation fuels from renewable sources via microbial fermentation. Central to this challenge is the design of biocatalysts that can efficiently convert cheap lignocellulosic raw materials into liquid fuels. Owing to the wealth of genetic and metabolic knowledge associated with Escherichia coli, this bacterium is the most convenient starting point for engineering microbial catalysts for biofuel production. Here, we review the range of liquid fuels that can be produced in E. coli and discuss the underlying biochemistry that enables these metabolic products. The fundamental and technological challenges encountered in the development of efficient fermentation processes for biofuel production are highlighted. The example of biodiesel is a particularly illustrative case study and is therefore discussed in detail.

    View details for DOI 10.1146/annurev-genet-102209-163440

    View details for Web of Science ID 000286042600003

    View details for PubMedID 20822440

  • The Biochemical Basis for Stereochemical Control in Polyketide Biosynthesis JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Valenzano, C. R., Lawson, R. J., Chen, A. Y., Khosla, C., Cane, D. E. 2009; 131 (51): 18501-18511

    Abstract

    One of the most striking features of complex polyketides is the presence of numerous methyl- and hydroxyl-bearing stereogenic centers. To investigate the biochemical basis for the control of polyketide stereochemistry and to establish the timing and mechanism of the epimerization at methyl-bearing centers, a series of incubations was carried out using reconstituted components from a variety of modular polyketide synthases. In all cases the stereochemistry of the product was directly correlated with the intrinsic stereospecificity of the ketoreductase domain, independent of the particular chain elongation domains that were used, thereby establishing that methyl group epimerization, when it does occur, takes place after ketosynthase-catalyzed chain elongation. The finding that there were only minor differences in the rates of product formation observed for parallel incubations using an epimerizing ketoreductase domain and the nonepimerizing ketoreductase domain supports the proposal that the epimerization is catalyzed by the ketoreductase domain itself.

    View details for DOI 10.1021/ja908296m

    View details for Web of Science ID 000273615800063

    View details for PubMedID 19928853

  • In Vivo and In Vitro Analysis of the Hedamycin Polyketide Synthase CHEMISTRY & BIOLOGY Das, A., Khosla, C. 2009; 16 (11): 1197-1207

    Abstract

    Hedamycin is an antitumor polyketide antibiotic with unusual biosynthetic features. Earlier sequence analysis of the hedamycin biosynthetic gene cluster implied a role for type I and type II polyketide synthases (PKSs). We demonstrate that the hedamycin minimal PKS can synthesize a dodecaketide backbone. The ketosynthase (KS) subunit of this PKS has specificity for both type I and type II acyl carrier proteins (ACPs) with which it collaborates during chain initiation and chain elongation, respectively. The KS receives a C(6) primer unit from the terminal ACP domain of HedU (a type I PKS protein) directly and subsequently interacts with the ACP domain of HedE (a type II PKS protein) during the process of chain elongation. HedE is a bifunctional protein with both ACP and aromatase activity. Its aromatase domain can modulate the chain length specificity of the minimal PKS. Chain length can also be influenced by HedA, the C-9 ketoreductase. While co-expression of the hedamycin minimal PKS and a chain-initiation module from the R1128 PKS yields an isobutyryl-primed decaketide, the orthologous PKS subunits from the hedamycin gene cluster itself are unable to prime the minimal PKS with a nonacetyl starter unit. Our findings provide new insights into the mechanism of chain initiation and elongation by type II PKSs.

    View details for DOI 10.1016/j.chembiol.2009.11.005

    View details for Web of Science ID 000273207900013

    View details for PubMedID 19942143

  • Structures and Mechanisms of Polyketide Synthases JOURNAL OF ORGANIC CHEMISTRY Khosla, C. 2009; 74 (17): 6416-6420

    Abstract

    Nearly a quarter-century ago, the advent of molecular genetic tools in the field of natural product biosynthesis led to the remarkable revelation that the genes responsible for the biosynthesis, regulation, and self-resistance of complex polyketide antibiotics were clustered in the genomes of the bacteria that produced these compounds. This in turn facilitated rapid cloning and sequencing of genes encoding a number of polyketide synthases (PKSs). By now, it is abundantly clear that, notwithstanding extraordinary architectural and biocatalytic diversity, all PKSs are evolutionarily related enzyme assemblies. As such, understanding the molecular logic for the biosynthesis of literally thousands of amazing polyketide natural products made by nature can benefit enormously from detailed investigations into a few "model systems". For nearly the past two decades, our laboratory has focused its efforts on two such PKSs. One of them synthesizes two polyketides in approximately equal ratios, SEK4 and SEK4b, and both shunt products from the pathway that leads to the biosynthesis of the pigmented antibiotic actinorhodin. The other synthesizes 6-deoxyerythronolide B, the first isolable intermediate in the biosynthetic pathway for the widely used antibacterial agent erythromycin. Our present-day knowledge of the structures and mechanisms of these two PKSs is summarized here.

    View details for DOI 10.1021/jo9012089

    View details for Web of Science ID 000269257800002

    View details for PubMedID 19711990

  • Noninflammatory Gluten Peptide Analogs as Biomarkers for Celiac Sprue CHEMISTRY & BIOLOGY Bethune, M. T., Crespo-Bosque, M., Bergseng, E., Mazumdar, K., Doyle, L., Sestak, K., Sollid, L. M., Khosla, C. 2009; 16 (8): 868-881

    Abstract

    New tools are needed for managing celiac sprue, a lifelong immune disease of the small intestine. Ongoing drug trials are also prompting a search for noninvasive biomarkers of gluten-induced intestinal change. We have synthesized and characterized noninflammatory gluten peptide analogs in which key Gln residues are replaced by Asn or His. Like their proinflammatory counterparts, these biomarkers are resistant to gastrointestinal proteases, susceptible to glutenases, and permeable across enterocyte barriers. Unlike gluten peptides, however, they are not appreciably recognized by transglutaminase, HLA-DQ2, or disease-specific T cells. In vitro and animal studies show that the biomarkers can detect intestinal permeability changes as well as glutenase-catalyzed gastric detoxification of gluten. Accordingly, controlled clinical studies are warranted to evaluate the use of these peptides as probes for abnormal intestinal permeability in celiac patients and for glutenase efficacy in clinical trials and practice.

    View details for DOI 10.1016/j.chembiol.2009.07.009

    View details for Web of Science ID 000269718200011

    View details for PubMedID 19716477

  • Modular Biocatalysts AICHE JOURNAL Khosla, C. 2009; 55 (8): 1926-1929

    View details for DOI 10.1002/aic.11981

    View details for Web of Science ID 000268413800002

  • A Food-Grade Enzyme Preparation with Modest Gluten Detoxification Properties PLOS ONE Ehren, J., Moron, B., Martin, E., Bethune, M. T., Gray, G. M., Khosla, C. 2009; 4 (7)

    Abstract

    Celiac sprue is a life-long disease characterized by an intestinal inflammatory response to dietary gluten. A gluten-free diet is an effective treatment for most patients, but accidental ingestion of gluten is common, leading to incomplete recovery or relapse. Food-grade proteases capable of detoxifying moderate quantities of dietary gluten could mitigate this problem.We evaluated the gluten detoxification properties of two food-grade enzymes, aspergillopepsin (ASP) from Aspergillus niger and dipeptidyl peptidase IV (DPPIV) from Aspergillus oryzae. The ability of each enzyme to hydrolyze gluten was tested against synthetic gluten peptides, a recombinant gluten protein, and simulated gastric digests of whole gluten and whole-wheat bread. Reaction products were analyzed by mass spectrometry, HPLC, ELISA with a monoclonal antibody that recognizes an immunodominant gluten epitope, and a T cell proliferation assay.ASP markedly enhanced gluten digestion relative to pepsin, and cleaved recombinant alpha2-gliadin at multiple sites in a non-specific manner. When used alone, neither ASP nor DPPIV efficiently cleaved synthetic immunotoxic gluten peptides. This lack of specificity for gluten was especially evident in the presence of casein, a competing dietary protein. However, supplementation of ASP with DPPIV enabled detoxification of moderate amounts of gluten in the presence of excess casein and in whole-wheat bread. ASP was also effective at enhancing the gluten-detoxifying efficacy of cysteine endoprotease EP-B2 under simulated gastric conditions.Clinical studies are warranted to evaluate whether a fixed dose ratio combination of ASP and DPPIV can provide near-term relief for celiac patients suffering from inadvertent gluten exposure. Due to its markedly greater hydrolytic activity against gluten than endogenous pepsin, food-grade ASP may also augment the activity of therapeutically relevant doses of glutenases such as EP-B2 and certain prolyl endopeptidases.

    View details for DOI 10.1371/journal.pone.0006313

    View details for Web of Science ID 000268147200010

    View details for PubMedID 19621078

  • Interferon-gamma Released by Gluten-Stimulated Celiac Disease-Specific Intestinal T Cells Enhances the Transepithelial Flux of Gluten Peptides JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Bethune, M. T., Siegel, M., Howles-Banerji, S., Khosla, C. 2009; 329 (2): 657-668

    Abstract

    Celiac sprue is a T-cell-mediated enteropathy elicited in genetically susceptible individuals by dietary gluten proteins. To initiate and propagate inflammation, proteolytically resistant gluten peptides must be translocated across the small intestinal epithelium and presented to DQ2-restricted T cells, but the effectors enabling this translocation under normal and inflammatory conditions are not well understood. We demonstrate that a fluorescently labeled antigenic 33-mer gluten peptide is translocated intact across a T84 cultured epithelial cell monolayer and that preincubation of the monolayer with media from gluten-stimulated, celiac patient-derived intestinal T cells enhances the apical-to-basolateral flux of this peptide in a dose-dependent, saturable manner. The permeability-enhancing activity of activated T-cell media is inhibited by blocking antibodies against either interferon-gamma or its receptor and is recapitulated using recombinant interferon-gamma. At saturating levels of interferon-gamma, activated T-cell media does not further increase transepithelial peptide flux, indicating the primacy of interferon-gamma as an effector of increased epithelial permeability during inflammation. Reducing the assay temperature to 4 degrees C reverses the effect of interferon-gamma but does not reduce basal peptide flux occurring in the absence of interferon-gamma, suggesting active transcellular transport of intact peptides is increased during inflammation. A panel of disease-relevant gluten peptides exhibited an inverse correlation between size and transepithelial flux but no apparent sequence constraints. Anti-interferon-gamma therapy may mitigate the vicious cycle of gluten-induced interferon-gamma secretion and interferon-gamma-mediated enhancement of gluten peptide flux but is unlikely to prevent translocation of gluten peptides in the absence of inflammatory conditions.

    View details for DOI 10.1124/jpet.108.148007

    View details for Web of Science ID 000265444000029

    View details for PubMedID 19218531

  • Biosynthesis of Aromatic Polyketides in Bacteria ACCOUNTS OF CHEMICAL RESEARCH Das, A., Khosla, C. 2009; 42 (5): 631-639

    Abstract

    Natural products, produced chiefly by microorganisms and plants, can be large and structurally complex molecules. These molecules are manufactured by cellular assembly lines, in which enzymes construct the molecules in a stepwise fashion. The means by which enzymes interact and work together in a modular fashion to create diverse structural features has been an active area of research; the work has provided insight into the fine details of biosynthesis. A number of polycyclic aromatic natural products--including several noteworthy anticancer, antibacterial, antifungal, antiviral, antiparasitic, and other medicinally significant substances--are synthesized by polyketide synthases (PKSs) in soil-borne bacteria called actinomycetes. Concerted biosynthetic, enzymological, and structural biological investigations into these modular enzyme systems have yielded interesting mechanistic insights. A core module called the minimal PKS is responsible for synthesizing a highly reactive, protein-bound poly-beta-ketothioester chain. In the absence of other enzymes, the minimal PKS also catalyzes chain initiation and release, yielding an assortment of polycyclic aromatic compounds. In the presence of an initiation PKS module, polyketide backbones bearing additional alkyl, alkenyl, or aryl primer units are synthesized, whereas a range of auxiliary PKS enzymes and tailoring enzymes convert the product of the minimal PKS into the final natural product. In this Account, we summarize the knowledge that has been gained regarding this family of PKSs through recent investigations into the biosynthetic pathways of two natural products, actinorhodin and R1128 (A-D). We also discuss the practical relevance of these fundamental insights for the engineered biosynthesis of new polycyclic aromatic compounds. With a deeper understanding of the biosynthetic process in hand, we can assert control at various stages of molecular construction and thus introduce unnatural functional groups in the process. The metabolic engineer affords a number of new avenues for creating novel molecular structures that will likely have properties akin to their fully natural cousins.

    View details for DOI 10.1021/ar8002249

    View details for Web of Science ID 000266238500006

    View details for PubMedID 19292437

  • Revisiting the modularity of modular polyketide synthases CURRENT OPINION IN CHEMICAL BIOLOGY Khosla, C., Kapur, S., Cane, D. E. 2009; 13 (2): 135-143

    Abstract

    Modularity is a highly sought after feature in engineering design. A modular catalyst is a multi-component system whose parts can be predictably interchanged for functional flexibility and variety. Nearly two decades after the discovery of the first modular polyketide synthase (PKS), we critically assess PKS modularity in the face of a growing body of atomic structural and in vitro biochemical investigations. Both the architectural modularity and the functional modularity of this family of enzymatic assembly lines are reviewed, and the fundamental challenges that lie ahead for the rational exploitation of their full biosynthetic potential are discussed.

    View details for DOI 10.1016/j.cbpa.2008.12.018

    View details for Web of Science ID 000266345400002

    View details for PubMedID 19217343

  • Evidence for Transcriptional Regulation of the Glucose-6-Phosphate Transporter by HIF-1 alpha: Targeting G6PT with Mumbaistatin Analogs in Hypoxic Mesenchymal Stromal Cells STEM CELLS Lord-Dufour, S., Copland, I. B., Levros, L., Post, M., Das, A., Khosla, C., Galipeau, J., Rassart, E., Annabi, B. 2009; 27 (3): 489-497

    Abstract

    Mesenchymal stromal cell (MSC) markers are expressed on brain tumor-initiating cells involved in the development of hypoxic glioblastoma. Given that MSCs can survive hypoxia and that the glucose-6-phosphate transporter (G6PT) provides metabolic control that contributes to MSC mobilization and survival, we investigated the effects of low oxygen (1.2% O(2)) exposure on G6PT gene expression. We found that MSCs significantly expressed G6PT and the glucose-6-phosphatase catalytic subunit beta, whereas expression of the glucose-6-phosphatase catalytic subunit alpha and the islet-specific glucose-6-phosphatase catalytic subunit-related protein was low to undetectable. Analysis of the G6PT promoter sequence revealed potential binding sites for hypoxia inducible factor (HIF)-1alpha and for the aryl hydrocarbon receptor (AhR) and its dimerization partner, the AhR nuclear translocator (ARNT), AhR:ARNT. In agreement with this, hypoxia and the hypoxia mimetic cobalt chloride induced the expression of G6PT, vascular endothelial growth factor (VEGF), and HIF-1alpha. Gene silencing of HIF-1alpha prevented G6PT and VEGF induction in hypoxic MSCs whereas generation of cells stably expressing HIF-1alpha resulted in increased endogenous G6PT gene expression. A semisynthetic analog of the polyketide mumbaistatin, a potent G6PT inhibitor, specifically reduced MSC-HIF-1alpha cell survival. Collectively, our data suggest that G6PT may account for the metabolic flexibility that enables MSCs to survive under conditions characterized by hypoxia and could be specifically targeted within developing tumors.

    View details for DOI 10.1634/stemcells.2008-0855

    View details for Web of Science ID 000264706900001

    View details for PubMedID 19074414

  • Protein engineering of improved prolyl endopeptidases for celiac sprue therapy PROTEIN ENGINEERING DESIGN & SELECTION Ehren, J., Govindarajan, S., Moron, B., Minshull, J., Khosla, C. 2008; 21 (12): 699-707

    Abstract

    Due to their unique ability to cleave immunotoxic gluten peptides endoproteolytically, prolyl endopeptidases (PEPs) are attractive oral therapeutic candidates for protecting celiac sprue patients from the toxic effects of dietary gluten. Enhancing the activity and stability of PEPs under gastric conditions (low pH, high pepsin concentration) is a challenge for protein engineers. Using a combination of sequence- and structure-based approaches together with machine learning algorithms, we have identified improved variants of the Sphingomonas capsulata PEP, a target of clinical relevance. Through two rounds of iterative mutagenesis and analysis, variants with as much as 20% enhanced specific activity at pH 4.5 and 200-fold greater resistance to pepsin were identified. Our results vividly reinforce the concept that conservative changes in proteins, especially in hydrophobic residues within tightly packed regions, can profoundly influence protein structure and function in ways that are difficult to predict entirely from first principles and must therefore be optimized through iterative design and analytical cycles. Incubation with whole wheat bread under simulated gastric conditions also suggests that some variants have pharmacologically significant improvements in gluten detoxification activity.

    View details for DOI 10.1093/protein/gzn050

    View details for Web of Science ID 000260979000002

    View details for PubMedID 18836204

  • Overproduction of free fatty acids in E. coli: Implications for biodiesel production METABOLIC ENGINEERING Lu, X., Vora, H., Khosla, C. 2008; 10 (6): 333-339

    Abstract

    Whereas microbial fermentation processes for producing ethanol and related alcohol biofuels are well established, biodiesel (methyl esters of fatty acids) is exclusively derived from plant oils. Slow cycle times for engineering oilseed metabolism and the excessive accumulation of glycerol as a byproduct are two major drawbacks of deriving biodiesel from plants. Although most bacteria produce fatty acids as cell envelope precursors, the biosynthesis of fatty acids is tightly regulated at multiple levels. By introducing four distinct genetic changes into the E. coli genome, we have engineered an efficient producer of fatty acids. Under fed-batch, defined media fermentation conditions, 2.5 g/L fatty acids were produced by this metabolically engineered E. coli strain, with a specific productivity of 0.024 g/h/g dry cell mass and a peak conversion efficiency of 4.8% of the carbon source into fatty acid products. At least 50% of the fatty acids produced were present in the free acid form.

    View details for DOI 10.1016/j.ymben.2008.08.006

    View details for Web of Science ID 000261946000006

    View details for PubMedID 18812230

  • Tissue transgluaminase 2 expression in meningiomas JOURNAL OF NEURO-ONCOLOGY Yuan, L., Behdad, A., Siegel, M., Khosla, C., Higashikubo, R., Rich, K. M. 2008; 90 (2): 125-132

    Abstract

    Meningiomas are common intracranial tumors that occur in extra-axial locations, most often over the cerebral convexities or along the skull-base. Although often histologically benign these tumors frequently present challenging clinical problems. Primary clinical management of patients with symptomatic tumors is surgical resection. Radiation treatment may arrest growth or delay recurrence of these tumors, however, meningioma cells are generally resistant to apoptosis after treatment with radiation. Tumor cells are known to alter their expression of proteins that interact in the ECM to provide signals important in tumor progression. One such protein, fibronectin, is expressed in elevated levels in the ECM in a number of tumors including meningiomas. We recently reported that levels of both extracellular fibronectin and tissue transglutaminase 2 (TG2) were increased in glioblastomas. We examined the expression of fibronectin and its association TG2 in meningiomas. Both fibronectin and TG2 were strongly expressed in all meningiomas studied. TG2 activity was markedly elevated in meningiomas, and TG2 was found to co-localize with fibronectin. Treatment of meningiomas with the small molecule TG2 inhibitor, KCC009, inhibited the binding of TG2 to fibronectin and blocked disposition of linear strands of fibronectin in the ECM. KCC009 treatment promoted apoptosis and enhanced radiation sensitivity both in cultured IOMM-Lee meningioma cells and in meningioma tumor explants. These findings support a potential protective role for TG2 in meningiomas.

    View details for DOI 10.1007/s11060-008-9642-1

    View details for Web of Science ID 000259672100001

    View details for PubMedID 18587533

  • Stereospecificity of ketoreductase domains 1 and 2 of the tylactone modular polyketide synthase JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Castonguay, R., Valenzano, C. R., Chen, A. Y., Keatinge-Clay, A., Khosla, C., Cane, D. E. 2008; 130 (35): 11598-?

    Abstract

    Tylactone synthase (TYLS) is a modular polyketide synthase that catalyzes the formation of tylactone (1), the parent aglycone precursor of the macrolide antibiotic tylosin. TYLS modules 1 and 2 are responsible for the generation of antidiketide and triketide intermediates, respectively, each bound to an acyl carrier protein (ACP) domain. Each module harbors a ketoreductase (KR) domain. The stereospecificity of TYLS KR1 and TYLS KR2 has been determined by incubating each of the recombinant ketoreductase domains with reconstituted ketosynthase-acyltransferase [KS][AT] and ACP domains from the 6-deoxyerythronolide B synthase (DEBS) in the presence of the N-acetylcysteamine thioester of syn-(2S,3R)-2-methyl-3-hydroxypentanoate (6), methylmalonyl-CoA, and NADPH resulting in the exclusive formation of the ACP-bound (2R,3R,4S,5R)-2,4-methyl-3,5-dihydroxyhepanoyl triketide, as established by GC-MS analysis of the TMS ether of the derived triketide lactone 7. Both TYLS KR1 and KR2 therefore catalyze the stereospecific reduction of the 2-methyl-3-ketoacyl-ACP substrate from the re-face, with specificity for the reduction of the (2R)-methyl (D) diastereomer. The dehydration that is catalyzed by the dehydratase (DH) domains of TYLS module 2 to give the unsaturated (2E,4S,5R)-2,4-dimethyl-5-hydroxyhept-2-enoyl-ACP2 is therefore a syn elimination of water.

    View details for DOI 10.1021/ja804453p

    View details for Web of Science ID 000258792000018

    View details for PubMedID 18693734

  • Biochemistry - Fit for an enzyme NATURE Kapur, S., Khosla, C. 2008; 454 (7206): 832-833

    View details for DOI 10.1038/454832a

    View details for Web of Science ID 000258398600019

    View details for PubMedID 18704072

  • Toward the Assessment of Food Toxicity for Celiac Patients: Characterization of Monoclonal Antibodies to a Main Immunogenic Gluten Peptide PLOS ONE Moron, B., Bethune, M. T., Comino, I., Manyani, H., Ferragud, M., Lopez, M. C., Cebolla, A., Khosla, C., Sousa, C. 2008; 3 (5)

    Abstract

    Celiac disease is a permanent intolerance to gluten prolamins from wheat, barley, rye and, in some patients, oats. Partially digested gluten peptides produced in the digestive tract cause inflammation of the small intestine. High throughput, immune-based assays using monoclonal antibodies specific for these immunotoxic peptides would facilitate their detection in food and enable monitoring of their enzymatic detoxification. Two monoclonal antibodies, G12 and A1, were developed against a highly immunotoxic 33-mer peptide. The potential of each antibody for quantifying food toxicity for celiac patients was studied.Epitope preferences of G12 and A1 antibodies were determined by ELISA with gluten-derived peptide variants of recombinant, synthetic or enzymatic origin.The recognition sequences of G12 and A1 antibodies were hexameric and heptameric epitopes, respectively. Although G12 affinity for the 33-mer was superior to A1, the sensitivity for gluten detection was higher for A1. This observation correlated to the higher number of A1 epitopes found in prolamins than G12 epitopes. Activation of T cell from gluten digested by glutenases decreased equivalently to the detection of intact peptides by A1 antibody. Peptide recognition of A1 included gliadin peptides involved in the both the adaptive and innate immunological response in celiac disease.The sensitivity and epitope preferences of the A1 antibody resulted to be useful to detect gluten relevant peptides to infer the potential toxicity of food for celiac patients as well as to monitor peptide modifications by transglutaminase 2 or glutenases.

    View details for DOI 10.1371/journal.pone.0002294

    View details for Web of Science ID 000262268500033

    View details for PubMedID 18509534

  • Mechanism based protein crosslinking of domains from the 6-deoxyerythronolide B synthase BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Kapur, S., Worthington, A., Tang, Y., Cane, D. E., Burkart, M. D., Khosla, C. 2008; 18 (10): 3034-3038

    Abstract

    The critical role of protein-protein interactions in the chemistry of polyketide synthases is well established. However, the transient and weak nature of these interactions, in particular those involving the acyl carrier protein (ACP), has hindered efforts to structurally characterize these interactions. We describe a chemo-enzymatic approach that crosslinks the active sites of ACP and their cognate ketosynthase (KS) domains, resulting in the formation of a stable covalent adduct. This process is driven by specific protein-protein interactions between KS and ACP domains. Suitable manipulation of the reaction conditions enabled complete crosslinking of a representative KS and ACP, allowing isolation of a stable, conformationally constrained adduct suitable for high-resolution structural analysis.

    View details for DOI 10.1016/j.bmcl.2008.01.073

    View details for Web of Science ID 000255954000004

    View details for PubMedID 18243693

  • Transepithelial Transport and Enzymatic Detoxification of Gluten in Gluten-Sensitive Rhesus Macaques PLOS ONE Bethune, M. T., Ribka, E., Khosla, C., Sestak, K. 2008; 3 (3)

    Abstract

    In a previous report, we characterized a condition of gluten sensitivity in juvenile rhesus macaques that is similar in many respects to the human condition of gluten sensitivity, celiac disease. This animal model of gluten sensitivity may therefore be useful toward studying both the pathogenesis and the treatment of celiac disease. Here, we perform two pilot experiments to demonstrate the potential utility of this model for studying intestinal permeability toward an immunotoxic gluten peptide and pharmacological detoxification of gluten in vivo by an oral enzyme drug candidate.Intestinal permeability was investigated in age-matched gluten-sensitive and control macaques by using mass spectrometry to detect and quantify an orally dosed, isotope labeled 33-mer gluten peptide delivered across the intestinal epithelium to the plasma. The protective effect of a therapeutically promising oral protease, EP-B2, was evaluated in a gluten-sensitive macaque by administering a daily gluten challenge with or without EP-B2 supplementation. ELISA-based antibody assays and blinded clinical evaluations of this macaque and of an age-matched control were conducted to assess responses to gluten.Labeled 33-mer peptide was detected in the plasma of a gluten-sensitive macaque, both in remission and during active disease, but not in the plasma of healthy controls. Administration of EP-B2, but not vehicle, prevented clinical relapse in response to a dietary gluten challenge. Unexpectedly, a marked increase in anti-gliadin (IgG and IgA) and anti-transglutaminase (IgG) antibodies was observed during the EP-B2 treatment phase.Gluten-sensitive rhesus macaques may be an attractive resource for investigating important aspects of celiac disease, including enhanced intestinal permeability and pharmacology of oral enzyme drug candidates. Orally dosed EP-B2 exerts a protective effect against ingested gluten. Limited data suggest that enhanced permeability of short gluten peptides generated by gastrically active glutenases may trigger an elevated antibody response, but that these antibodies are not necessarily causative of clinical illness.

    View details for DOI 10.1371/journal.pone.0001857

    View details for Web of Science ID 000260762400018

    View details for PubMedID 18365012

  • Extracellular Transglutaminase 2 Is Catalytically Inactive, but Is Transiently Activated upon Tissue Injury PLOS ONE Siegel, M., Strnad, P., Watts, R. E., Choi, K., Jabri, B., Omary, M. B., Khosla, C. 2008; 3 (3)

    Abstract

    Transglutaminase 2 (TG2) is a multifunctional mammalian protein with transamidase and signaling properties. Using selective TG2 inhibitors and tagged nucleophilic amine substrates, we show that the majority of extracellular TG2 is inactive under normal physiological conditions in cell culture and in vivo. However, abundant TG2 activity was detected around the wound in a standard cultured fibroblast scratch assay. To demonstrate wounding-induced activation of TG2 in vivo, the toll-like receptor 3 ligand, polyinosinic-polycytidylic acid (poly(I:C)), was injected in mice to trigger small intestinal injury. Although no TG2 activity was detected in vehicle-treated mice, acute poly(I:C) injury resulted in rapid TG2 activation in the small intestinal mucosa. Our findings provide a new basis for understanding the role of TG2 in physiology and disease.

    View details for DOI 10.1371/journal.pone.0001861

    View details for Web of Science ID 000260762400022

    View details for PubMedID 18365016

  • Evolution of polyketide synthases in bacteria PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ridley, C. P., Lee, H. Y., Khosla, C. 2008; 105 (12): 4595-4600

    Abstract

    The emergence of resistant strains of human pathogens to current antibiotics, along with the demonstrated ability of polyketides as antimicrobial agents, provides strong motivation for understanding how polyketide antibiotics have evolved and diversified in nature. Insights into how bacterial polyketide synthases (PKSs) acquire new metabolic capabilities can guide future laboratory efforts in generating the next generation of polyketide antibiotics. Here, we examine phylogenetic and structural evidence to glean answers to two general questions regarding PKS evolution. How did the exceptionally diverse chemistry of present-day PKSs evolve? And what are the take-home messages for the biosynthetic engineer?

    View details for Web of Science ID 000254772700014

    View details for PubMedID 18250311

  • A Non-Human Primate Model for Gluten Sensitivity PLOS ONE Bethune, M. T., Borda, J. T., Ribka, E., Liu, M., Phillippi-Falkenstein, K., Jandacek, R. J., Doxiadis, G. G., Gray, G. M., Khosla, C., Sestak, K. 2008; 3 (2)

    Abstract

    Gluten sensitivity is widespread among humans. For example, in celiac disease patients, an inflammatory response to dietary gluten leads to enteropathy, malabsorption, circulating antibodies against gluten and transglutaminase 2, and clinical symptoms such as diarrhea. There is a growing need in fundamental and translational research for animal models that exhibit aspects of human gluten sensitivity.Using ELISA-based antibody assays, we screened a population of captive rhesus macaques with chronic diarrhea of non-infectious origin to estimate the incidence of gluten sensitivity. A selected animal with elevated anti-gliadin antibodies and a matched control were extensively studied through alternating periods of gluten-free diet and gluten challenge. Blinded clinical and histological evaluations were conducted to seek evidence for gluten sensitivity.When fed with a gluten-containing diet, gluten-sensitive macaques showed signs and symptoms of celiac disease including chronic diarrhea, malabsorptive steatorrhea, intestinal lesions and anti-gliadin antibodies. A gluten-free diet reversed these clinical, histological and serological features, while reintroduction of dietary gluten caused rapid relapse.Gluten-sensitive rhesus macaques may be an attractive resource for investigating both the pathogenesis and the treatment of celiac disease.

    View details for DOI 10.1371/journal.pone.0001614

    View details for Web of Science ID 000260586400007

    View details for PubMedID 18286171

  • Parallels between pathogens and gluten peptides in celiac sprue PLOS PATHOGENS Bethune, M. T., Khosla, C. 2008; 4 (2)

    Abstract

    Pathogens are exogenous agents capable of causing disease in susceptible organisms. In celiac sprue, a disease triggered by partially hydrolyzed gluten peptides in the small intestine, the offending immunotoxins cannot replicate, but otherwise have many hallmarks of classical pathogens. First, dietary gluten and its peptide metabolites are ubiquitous components of the modern diet, yet only a small, genetically susceptible fraction of the human population contracts celiac sprue. Second, immunotoxic gluten peptides have certain unusual structural features that allow them to survive the harsh proteolytic conditions of the gastrointestinal tract and thereby interact extensively with the mucosal lining of the small intestine. Third, they invade across epithelial barriers intact to access the underlying gut-associated lymphoid tissue. Fourth, they possess recognition sequences for selective modification by an endogenous enzyme, transglutaminase 2, allowing for in situ activation to a more immunotoxic form via host subversion. Fifth, they precipitate a T cell-mediated immune reaction comprising both innate and adaptive responses that causes chronic inflammation of the small intestine. Sixth, complete elimination of immunotoxic gluten peptides from the celiac diet results in remission, whereas reintroduction of gluten in the diet causes relapse. Therefore, in analogy with antibiotics, orally administered proteases that reduce the host's exposure to the immunotoxin by accelerating gluten peptide destruction have considerable therapeutic potential. Last but not least, notwithstanding the power of in vitro methods to reconstitute the essence of the immune response to gluten in a celiac patient, animal models for the disease, while elusive, are likely to yield fundamentally new systems-level insights.

    View details for DOI 10.1371/journal.ppat.0040034

    View details for Web of Science ID 000255415900002

    View details for PubMedID 18425213

  • Transglutaminase 2 undergoes a large conformational change upon activation PLOS BIOLOGY Pinkas, D. M., Strop, P., Brunger, A. T., Khosla, C. 2007; 5 (12): 2788-2796

    Abstract

    Human transglutaminase 2 (TG2), a member of a large family of enzymes that catalyze protein crosslinking, plays an important role in the extracellular matrix biology of many tissues and is implicated in the gluten-induced pathogenesis of celiac sprue. Although vertebrate transglutaminases have been studied extensively, thus far all structurally characterized members of this family have been crystallized in conformations with inaccessible active sites. We have trapped human TG2 in complex with an inhibitor that mimics inflammatory gluten peptide substrates and have solved, at 2-A resolution, its x-ray crystal structure. The inhibitor stabilizes TG2 in an extended conformation that is dramatically different from earlier transglutaminase structures. The active site is exposed, revealing that catalysis takes place in a tunnel, bridged by two tryptophan residues that separate acyl-donor from acyl-acceptor and stabilize the tetrahedral reaction intermediates. Site-directed mutagenesis was used to investigate the acyl-acceptor side of the tunnel, yielding mutants with a marked increase in preference for hydrolysis over transamidation. By providing the ability to visualize this activated conformer, our results create a foundation for understanding the catalytic as well as the non-catalytic roles of TG2 in biology, and for dissecting the process by which the autoantibody response to TG2 is induced in celiac sprue patients.

    View details for DOI 10.1371/journal.pbio.0050327

    View details for Web of Science ID 000251874900010

    View details for PubMedID 18092889

  • Stereospecificity of ketoreductase domains of the 6-deoxyerythronolide B synthase JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Castonguay, R., He, W., Chen, A. Y., Khosla, C., Cane, D. E. 2007; 129 (44): 13758-13769

    Abstract

    6-Deoxyerythronolide B synthase (DEBS) is a modular polyketide synthase (PKS) responsible for the biosynthesis of 6-dEB (1), the parent aglycone of the broad spectrum macrolide antibiotic erythromycin. Individual DEBS modules, which contain the catalytic domains necessary for each step of polyketide chain elongation and chemical modification, can be deconstructed into constituent domains. To better understand the intrinsic stereospecificity of the ketoreductase (KR) domains, an in vitro reconstituted system has been developed involving combinations of ketosynthase (KS)-acyl transferase (AT) didomains with acyl-carrier protein (ACP) and KR domains from different DEBS modules. Incubations with (2S,3R)-2-methyl-3-hydroxypentanoic acid N-acetylcysteamine thioester (2) and methylmalonyl-CoA plus NADPH result in formation of a reduced, ACP-bound triketide that is converted to the corresponding triketide lactone 4 by either base- or enzyme-catalyzed hydrolysis/cyclization. A sensitive and robust GC-MS technique has been developed to assign the stereochemistry of the resulting triketide lactones, on the basis of direct comparison with synthetic standards of each of the four possible diasteromers 4a-4d. Using the [KS][AT] didomains from either DEBS module 3 or module 6 in combination with KR domains from modules 2 or 6 gave in all cases exclusively (2R,3S,4R,5R)-3,5-dihydroxy-2,4-dimethyl-n-heptanoic acid-delta-lactone (4a). The same product was also generated by a chimeric module in which [KS3][AT3] was fused to [KR5][ACP5] and the DEBS thioesterase [TE] domain. Reductive quenching of the ACP-bound 2-methyl-3-ketoacyl triketide intermediate with sodium borohydride confirmed that in each case the triketide intermediate carried only an unepimerized d-2-methyl group. The results confirm the predicted stereospecificity of the individual KR domains, while revealing an unexpected configurational stability of the ACP-bound 2-methyl-3-ketoacyl thioester intermediate. The methodology should be applicable to the study of any combination of heterologous [KS][AT] and [KR] domains.

    View details for DOI 10.1021/ja0753290

    View details for Web of Science ID 000250819200064

    View details for PubMedID 17918944

  • Cyclic and dimeric gluten peptide analogues inhibiting DQ2-mediated antigen presentation in celiac disease BIOORGANIC & MEDICINAL CHEMISTRY Xia, J., Bergseng, E., Fleckenstein, B., Siegel, M., Kim, C., Khosla, C., Sollid, L. M. 2007; 15 (20): 6565-6573

    Abstract

    Celiac disease is an immune mediated enteropathy elicited by gluten ingestion. The disorder has a strong association with HLA-DQ2. This HLA molecule is involved in the disease pathogenesis by presenting gluten peptides to T cells. Blocking the peptide-binding site of DQ2 may be a way to treat celiac disease. In this study, two types of peptide analogues, modeled after natural gluten antigens, were studied as DQ2 blockers. (a) Cyclic peptides. Cyclic peptides containing the DQ2-alphaI gliadin epitope LQPFPQPELPY were synthesized with flanking cysteine residues introduced and subsequently crosslinked via a disulfide bond. Alternatively, cyclic peptides were prepared with stable polyethylene glycol bridges across internal lysine residues of modified antigenic peptides such as KQPFPEKELPY and LQLQPFPQPEKPYPQPEKPY. The effect of cyclization as well as the length of the spacer in the cyclic peptides on DQ2 binding and T cell recognition was analyzed. Inhibition of peptide-DQ2 recognition by the T cell receptor was observed in T cell proliferation assays. (b) Dimeric peptides. Previously we developed a new type of peptide blocker with much enhanced affinity for DQ2 by dimerizing LQLQPFPQPEKPYPQPELPY through the lysine side chains. Herein, the effect of linker length on both DQ2 binding and T cell inhibition was investigated. One dimeric peptide analogue with an intermediate linker length was found to be especially effective at inhibiting DQ2 mediated antigen presentation. The implications of these findings for the treatment of celiac disease are discussed.

    View details for DOI 10.1016/j.bmc.2007.07.001

    View details for Web of Science ID 000249713900008

    View details for PubMedID 17681795

  • Solution structure and proposed domain-domain recognition interface of an acyl carrier protein domain from a modular polyketide synthase PROTEIN SCIENCE Alekseyev, V. Y., Liu, C. W., Cane, D. E., Puglisi, J. D., Khosla, C. 2007; 16 (10): 2093-2107

    Abstract

    Polyketides are a medicinally important class of natural products. The architecture of modular polyketide synthases (PKSs), composed of multiple covalently linked domains grouped into modules, provides an attractive framework for engineering novel polyketide-producing assemblies. However, impaired domain-domain interactions can compromise the efficiency of engineered polyketide biosynthesis. To facilitate the study of these domain-domain interactions, we have used nuclear magnetic resonance (NMR) spectroscopy to determine the first solution structure of an acyl carrier protein (ACP) domain from a modular PKS, 6-deoxyerythronolide B synthase (DEBS). The tertiary fold of this 10-kD domain is a three-helical bundle; an additional short helix in the second loop also contributes to the core helical packing. Superposition of residues 14-94 of the ensemble on the mean structure yields an average atomic RMSD of 0.64 +/- 0.09 Angstrom for the backbone atoms (1.21 +/- 0.13 Angstrom for all non-hydrogen atoms). The three major helices superimpose with a backbone RMSD of 0.48 +/- 0.10 Angstrom (0.99 +/- 0.11 Angstrom for non-hydrogen atoms). Based on this solution structure, homology models were constructed for five other DEBS ACP domains. Comparison of their steric and electrostatic surfaces at the putative interaction interface (centered on helix II) suggests a model for protein-protein recognition of ACP domains, consistent with the previously observed specificity. Site-directed mutagenesis experiments indicate that two of the identified residues influence the specificity of ACP recognition.

    View details for DOI 10.1110/ps.073011407

    View details for Web of Science ID 000249692400001

    View details for PubMedID 17893358

  • Structure-based design of alpha-amido aldehyde containing gluten peptide analogues as modulators of HLA-DQ2 and transglutaminase 2 BIOORGANIC & MEDICINAL CHEMISTRY Siegel, M., Xia, J., Khosla, C. 2007; 15 (18): 6253-6261

    Abstract

    Complete, life-long exclusion of gluten containing foods from the diet is the only available treatment for celiac sprue, a widespread immune disease of the small intestine. Investigations into the molecular pathogenesis of celiac sprue have identified the major histocompatibility complex protein HLA-DQ2 and the multi-functional enzyme transglutaminase 2 as potential pharmacological targets. Based upon the crystal structure of HLA-DQ2, we rationally designed an aldehyde-functionalized, gluten peptide analogue as a tight-binding HLA-DQ2 ligand. Aldehyde-bearing gluten peptide analogues were also designed as high-affinity, reversible inhibitors of transglutaminase 2. By varying the side-chain length of the aldehyde-functionalized amino acid, we found that the optimal transglutaminase 2 inhibitor was 5 methylene units in length, 2 carbon atoms longer than its natural glutamine substrate.

    View details for DOI 10.1016/j.bmc.2007.06.020

    View details for Web of Science ID 000249194900027

    View details for PubMedID 17590341

  • A scaleable manufacturing process for pro-EP-B2, a cysteine protease from barley indicated for Celiac Sprue BIOTECHNOLOGY AND BIOENGINEERING Vora, H., McIntire, J., Kumar, P., Deshpande, M., Khosla, C. 2007; 98 (1): 177-185

    Abstract

    Celiac Sprue is an inflammatory disease of the small intestine triggered by ingestion of dietary gluten, a family of glutamine and proline rich proteins found in common foodgrains such as wheat, rye, and barley. One potential therapy for this lifelong disease anticipates using an oral protease to detoxify gluten in vivo. Recent studies have shown that EP-B2 (endoprotease B, isoform 2) from barley is a promising example of such a glutenase, thus warranting its large-scale production for animal safety and human clinical studies. Here we describe a scaleable fermentation, refolding and purification process for the production of gram to kilogram quantities of pro-EP-B2 (zymogen form of EP-B2) in a lyophilized form. A fed-batch E. coli fermentation system was developed that yields 0.3-0.5 g purified recombinant protein per liter culture volume. Intracellular degradation of pro-EP-B2 during the fermentation was overcome by manipulating the fermentation temperature and duration of protein expression. A simple refolding protocol was developed using a fast dilution method to refold the enzyme at concentrations greater than 0.5 mg/mL. Kinetic analysis showed that pro-EP-B2 refolding is a first-order reaction with an estimated rate constant of 0.15 h(-1). A lyophilization procedure was developed that yielded protein with 85% recoverable activity after 7 weeks of storage at room temperature. The process was successfully scaled up to 100 L with comparable purity and recovery.

    View details for DOI 10.1002/bit.21423

    View details for Web of Science ID 000248655700018

    View details for PubMedID 17385743

  • Combination enzyme therapy for gastric digestion of dietary gluten in patients with celiac sprue GASTROENTEROLOGY Gass, J., Bethune, M. T., Siegel, M., Spencer, A., Khosla, C. 2007; 133 (2): 472-480

    Abstract

    Celiac sprue is a multifactorial disease characterized by an inflammatory response to ingested gluten in the small intestine. Proteolytically resistant, proline- and glutamine-rich gluten peptides from wheat, rye, and barley persist in the intestinal lumen and elicit an immune response in genetically susceptible persons. We investigated a new combination enzyme product, consisting of a glutamine-specific endoprotease (EP-B2 from barley) and a prolyl endopeptidase (SC PEP from Sphingomonas capsulata), for its ability to digest gluten under gastric conditions.The ability of this combination enzyme to digest and detoxify whole-wheat bread gluten was investigated. In vitro and in vivo (rat) experimental systems were developed to simulate human gastric digestion, and the resulting material was analyzed by high-performance liquid chromatography, enzyme-linked immunoabsorbent assay, and patient-derived T-cell proliferation assays.The analysis revealed that EP-B2 extensively proteolyzes complex gluten proteins in bread, whereas SC PEP rapidly detoxifies the residual oligopeptide products of EP-B2 digestion. In vitro dose variation data suggests that an approximate 1:1 weight ratio of the 2 enzymes should maximize their synergistic potential. The efficacy of this 2-enzyme glutenase was verified in a rat model of gastric gluten digestion.By combining 2 enzymes with gastric activity and complementary substrate specificity, it should be possible to increase the safe threshold of ingested gluten, thereby ameliorating the burden of a highly restricted diet for patients with celiac sprue.

    View details for DOI 10.1053/j.gastro.2007.05.028

    View details for Web of Science ID 000248585600017

    View details for PubMedID 17681168

  • Transglutaminase 2 inhibitors and their therapeutic role in disease states PHARMACOLOGY & THERAPEUTICS Siegel, M., Khosla, C. 2007; 115 (2): 232-245

    Abstract

    Transglutaminase 2 (TG2) is a multi-domain, multi-functional enzyme that post-translationally modifies proteins by catalyzing the formation of intermolecular isopeptide bonds between glutamine and lysine side-chains. It plays a role in diverse biological functions, including extracellular matrix formation, integrin-mediated signaling, and signal transduction involving 7-transmembrane receptors. While some of the roles of TG2 under normal physiological conditions remain obscure, the protein is believed to participate in the pathogenesis of several unrelated diseases, including celiac sprue, neurodegenerative diseases, and certain types of cancer. A variety of small molecule and peptidomimetic inhibitors of the TG2 active site have been identified. Here, we summarize the biochemistry, biology, pharmacology and medicinal chemistry of human TG2.

    View details for DOI 10.1016/j.pharmthera.2007.05.003

    View details for Web of Science ID 000248657000004

    View details for PubMedID 17582505

  • Structure-activity relationships of semisynthetic mumbaistatin analogs BIOORGANIC & MEDICINAL CHEMISTRY Lee, T. S., Das, A., Khosla, C. 2007; 15 (15): 5207-5218

    Abstract

    Mumbaistatin (1), a new anthraquinone natural product, is one of the most potent known inhibitors of hepatic glucose-6-phosphate translocase, an important target for the treatment of type II diabetes. Its availability, however, has been limited due to its extremely low yield from the natural source. Starting from DMAC (5, 3,8-dihydroxyanthraquinone-2-carboxylic acid), a structurally related polyketide product of engineered biosynthesis, we developed a facile semisynthetic method that afforded a variety of mumbaistatin analogs within five steps. This work was facilitated by the initial development of a DMAC overproduction system. In addition to reinforcing the biological significance of the anthraquinone moiety of mumbaistatin, several semisynthetic analogs were found to have low micromolar potency against the translocase in vitro. Two of them were also active in glucose release assays from primary hepatocytes. The synergistic combination of biosynthesis and synthesis is a promising avenue for the discovery of new bioactive substances.

    View details for DOI 10.1016/j.bmc.2007.05.019

    View details for Web of Science ID 000247714900017

    View details for PubMedID 17524653

  • Structural and mechanistic analysis of protein interactions in module 3 of the 6-deoxyerythronolide B synthase CHEMISTRY & BIOLOGY Tang, Y., Chen, A. Y., Kim, C., Cane, D. E., Khosla, C. 2007; 14 (8): 931-943

    Abstract

    We report the 2.6 A X-ray crystal structure of a 190 kDa homodimeric fragment from module 3 of the 6-deoxyerthronolide B synthase covalently bound to the inhibitor cerulenin. The structure shows two well-organized interdomain linker regions in addition to the full-length ketosynthase (KS) and acyltransferase (AT) domains. Analysis of the substrate-binding site of the KS domain suggests that a loop region at the homodimer interface influences KS substrate specificity. We also describe a model for the interaction of the catalytic domains with the acyl carrier protein (ACP) domain. The ACP is proposed to dock within a deep cleft between the KS and AT domains, with interactions that span both the KS homodimer and AT domain. In conjunction with other recent data, our results provide atomic resolution pictures of several catalytically relevant protein interactions in this remarkable family of modular megasynthases.

    View details for DOI 10.1016/j.chembiol.2007.07.012

    View details for Web of Science ID 000249257800010

    View details for PubMedID 17719492

  • Structure-based dissociation of a type I polyketide synthase module CHEMISTRY & BIOLOGY Chen, A. Y., Cane, D. E., Khosla, C. 2007; 14 (7): 784-792

    Abstract

    Individual modules of modular polyketide synthases (PKSs) such as 6-deoxyerythronolide B synthase (DEBS) consist of conserved, covalently linked domains separated by unconserved intervening linker sequences. To better understand the protein-protein and enzyme-substrate interactions in modular catalysis, we have exploited recent structural insights to prepare stand-alone domains of selected DEBS modules. When combined in vitro, ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP) domains of DEBS module 3 catalyzed methylmalonyl transfer and diketide substrate elongation. When added to a minimal PKS, ketoreductase domains from DEBS modules 1, 2, and 6 showed specificity for the beta-ketoacylthioester substrate, but not for either the ACP domain carrying the polyketide substrate or the KS domain that synthesized the substrate. With insights into catalytic efficiency and specificity of PKS modules, our results provide guidelines for constructing optimal hybrid PKS systems.

    View details for DOI 10.1016/j.chembiol.2007.05.015

    View details for Web of Science ID 000248402400010

    View details for PubMedID 17656315

  • Enhancement of dietary protein digestion by conjugated bile acids GASTROENTEROLOGY Gass, J., Vora, H., Hofmann, A. F., Gray, G. M., Khosla, C. 2007; 133 (1): 16-23

    Abstract

    Conjugated bile acids promote absorption of dietary lipids by solubilizing them in mixed micelles. Bile acids are not considered to facilitate the digestion of other nutrients.The effect of conjugated bile acids on the rate of protein hydrolysis by trypsin and chymotrypsin was examined in vitro. Common dietary proteins and 2 bacterial glutenases (proposed oral therapies for celiac sprue) were proteolyzed in the absence or presence of a 10 mmol/L conjugated bile acid mixture, simulating human bile composition. Lipolysis products (monoolein) and fatty acid were also evaluated to simulate postprandial intestinal contents.Conjugated bile acids dramatically enhanced the proteolysis of several dietary proteins, including beta-lactoglobulin, bovine serum albumin, myoglobin, and a commercially available dietary protein supplement. For beta-lactoglobulin, a cow's milk allergen that is resistant to pepsin cleavage, bile acids enhanced its proteolysis by pancreatic proteases even after incubation under gastric conditions. Exposure of prolyl endopeptidases to bile acids made them more susceptible to pancreatic proteases under simulated intestinal conditions. The conjugated bile acid effect was most pronounced in the presence of dihydroxy bile acids and was observable at bile concentrations below the critical micellar concentration but to a much greater extent at concentrations above the critical micellar concentration.We propose that, in addition to promoting lipid absorption, conjugated bile acids affect the digestion and assimilation of dietary proteins by accelerating hydrolysis by pancreatic proteases. These findings have implications for intraluminal protein breakdown and assimilation in the upper small intestine.

    View details for DOI 10.1053/j.gastro.2007.04.008

    View details for Web of Science ID 000248055400007

    View details for PubMedID 17631126

  • Transglutaminase 2 inhibitor, KCC009, disrupts fibronectin assembly in the extracellular matrix and sensitizes orthotopic glioblastomas to chemotherapy ONCOGENE Yuan, L., Siegel, M., Choi, K., Khosla, C., Miller, C. R., Jackson, E. N., Piwnica-Worms, D., Rich, K. M. 2007; 26 (18): 2563-2573

    Abstract

    Transglutaminase 2 (TG2, a.k.a. tissue transglutaminase) belongs to a family of transglutaminase enzymes that stabilize proteins by affecting covalent crosslinking via formation of amide bonds. Cell surface TG2 is directly involved as an adhesive receptor in cell-extracellular matrix (ECM) interactions. Here, we show that TG2 activity is elevated in glioblastomas compared with non-neoplastic brain. Immunofluorescent studies showed increased staining of fibronectin colocalized with TG2 in the ECM in glioblastomas. In addition, small clusters of invading human glioblastoma cells present in non-neoplastic brain parenchyma secrete high levels of TG2 and fibronectin that distinguish them from normal brain stroma. Downregulation of TG2 in U87MG glioblastoma cells with RNAi demonstrated decreased assembly of fibronectin in the ECM. Treatment with KCC009 blocked the remodeling of fibronectin in the ECM in glioblastomas in both in vitro and in vivo studies. KCC009 treatment in mice harboring orthotopic glioblastomas (DBT-FG) sensitized the tumors to N,N'-bis(2-chloroethyl)-N-nitrosourea chemotherapy, as measured by reduced bioluminescence, increased apoptosis and prolonged survival. The ability of KCC009 to interfere with the permissive remodeling of fibronectin in the ECM in glioblastomas suggests a novel target to enhance sensitivity to chemotherapy directed not only at the tumor mass, but also invading glioblastoma cells.

    View details for DOI 10.1038/sj.onc.1210048

    View details for Web of Science ID 000245831200004

    View details for PubMedID 17099729

  • Antibiotic production from the ground up NATURE BIOTECHNOLOGY Katz, L., Khosla, C. 2007; 25 (4): 428-429

    View details for Web of Science ID 000245651800020

    View details for PubMedID 17420748

  • Transglutaminase 2 regulates mallory body inclusion formation and injury-associated liver enlargement GASTROENTEROLOGY Strnad, P., Harada, M., Siegel, M., Terkeltaub, R. A., Graham, R. M., Khosla, C., Omary, M. B. 2007; 132 (4): 1515-1526

    Abstract

    Mallory body (MB) inclusions are a characteristic feature of several liver disorders and share similarities with cytoplasmic inclusions observed in neural diseases and myopathies. MBs consist primarily of keratins 8 and 18 (K8/K18), require a K8-greater-than-K18 ratio for their formation, and contain glutamine-lysine cross-links generated by transglutaminase (TG). We hypothesized that protein transamidation is essential for MB formation.Because TG2 is the most abundant hepatocyte TG, we tested our hypothesis using TG2(-/-) and their wild-type counterpart mice fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), an established MB inducer. Keratin cross-linking was further examined using recombinant proteins or transgenic mice that overexpress K8 or K18.TG2(-/-) livers have markedly reduced TG2 activity as compared with TG2(+/+) livers. The DDC-fed TG2(-/-) mice have dramatic decreases in MB formation and liver hypertrophy response as contrasted with DDC-fed TG2(+/+) mice. Despite similar hepatocellular damage, TG2(-/-) mice had more gallstones, jaundice, and ductal proliferation than wild-type mice. Inhibition of MB formation in TG2(-/-) mice was associated with marked attenuation of ubiquitination and K8-containing protein cross-linking. MB formation and resolution paralleled the generation then disappearance of cross-linked K8, respectively. K8 is a preferential TG2 substrate when compared to K18, as examined in vitro or in DDC-fed transgenic mice that overexpress K8 or K18.We demonstrate an essential role for TG2 in determining injury-mediated liver enlargement and the necessity of K8 and TG2 for generating cross-linked keratins and MBs. The role of TG in inclusion formation might extend to nonkeratin intermediate filament protein-related diseases.

    View details for DOI 10.1053/j.gastro.2007.02.020

    View details for Web of Science ID 000246020900037

    View details for PubMedID 17408647

  • Substrate tolerance of module 6 of the epothilone synthetase BIOCHEMISTRY Tse, M. L., Watts, R. E., Khosla, C. 2007; 46 (11): 3385-3393

    Abstract

    The epothilone synthetase is a decamodular megasynthase responsible for the biosynthesis of a class of polyketide natural products with clinically promising antitumor activity. Recently, we developed a system comprised of modules 6-9 of the epothilone synthetase for the precursor-directed biosynthesis of epothilones in Escherichia coli [Boddy, C. N., Hotta, K., Tse, M. L., Watts, R. E., and Khosla, C. (2004) J. Am. Chem. Soc. 126, 7436-7437]. To systematically explore the biosynthetic potential of this system, we have now investigated the ability of the crucial first module in this engineered pathway, EpoD-M6, to accept, elongate, and process unnatural substrates. EpoD-M6 was expressed, purified, and demonstrated to accept both acyl-CoA and acylSNAC substrates. Of the substrates that were tested, octanoylSNAC and 3-octenoylSNAC proved to be excellent substrates in addition to the more complex natural substrate. Thus, this polyketide synthase module showed considerable tolerance, a feature that bodes well for the precursor-directed biosynthesis of epothilone analogues and related complex polyketides.

    View details for DOI 10.1021/bi0616448

    View details for Web of Science ID 000244854800047

    View details for PubMedID 17315981

  • Bioassay-Guided evolution of glycosylated macrolide antibiotics in Escherichia coli PLOS BIOLOGY Lee, H. Y., Khosla, C. 2007; 5 (2): 243-250

    Abstract

    Macrolide antibiotics such as erythromycin are clinically important polyketide natural products. We have engineered a recombinant strain of Escherichia coli that produces small but measurable quantities of the bioactive macrolide 6-deoxyerythromycin D. Bioassay-guided evolution of this strain led to the identification of an antibiotic-overproducing mutation in the mycarose biosynthesis and transfer pathway that was detectable via a colony-based screening assay. This high-throughput assay was then used to evolve second-generation mutants capable of enhanced precursor-directed biosynthesis of macrolide antibiotics. The availability of a screen for macrolide biosynthesis in E. coli offers a fundamentally new approach in dissecting modular megasynthase mechanisms as well as engineering antibiotics with novel pharmacological properties.

    View details for DOI 10.1371/journal.pbio.0050045

    View details for Web of Science ID 000245243200012

    View details for PubMedID 17298179

  • Synthesis and biological activity of novel pyranopyrones derived from engineered aromatic polyketides ACS CHEMICAL BIOLOGY Ridley, C. P., Khosla, C. 2007; 2 (2): 104-108

    Abstract

    The 4-hydroxy-2-pyrone moiety is commonly observed in polyketides generated via biosynthetic engineering of type II polyketide synthases. To explore the synthetic utility of these 2-pyrones, four engineered polyketides (mutactin, SEK4, SEK15, and SEK15b) were isolated from appropriate derivatives of Streptomyces coelicolor CH999. As a test case, we prepared nine novel pyranopyrones through condensation reactions with either citral, 1-cyclohexene-carboxaldehyde, or S-perillaldehyde. Synthetic tricyclic pyranopyrones with simple aromatic substituents are known to possess anticancer properties. We therefore investigated whether pyranopyrone derivatives of aromatic polyketides exhibited bioactivity in a panel of three cancer cell lines. Pyranopyrones generated from SEK4 had activity comparable to that of H10, a pyranopyrone with a 3-pyridyl substituent, whereas other analogues were less active. These results suggest that the diverse library of carbo- and heterocycles available through the genetic engineering of type II polyketide synthases can serve as useful building blocks to generate unique bioactive molecules.

    View details for DOI 10.1021/cb600382j

    View details for Web of Science ID 000244347600016

    View details for PubMedID 17256996

  • Prolyl endopeptidases CELLULAR AND MOLECULAR LIFE SCIENCES Gass, J., Khosla, C. 2007; 64 (3): 345-355

    Abstract

    This review describes the structure and function of prolyl endopeptidase (PEP) enzymes and how they are being evaluated as drug targets and therapeutic agents. The most well studied PEP family has a two-domain structure whose unique seven-blade beta-propeller domain works with the catalytic domain to hydrolyze the peptide bond on the carboxyl side of internal proline residues of an oligopeptide substrate. Structural and functional studies on this protease family have elucidated the mechanism for peptide entry between the two domains. Other structurally unrelated PEPs have been identified, but have not been studied in detail. Human PEP has been evaluated as a pharmacological target for neurological diseases due to its high brain concentration and ability to cleave neuropeptides in vitro. Recently, microbial PEPs have been studied as potential therapeutics for celiac sprue, an inflammatory disease of the small intestine triggered by proline-rich gluten.

    View details for DOI 10.1007/s00018-006-6317-y

    View details for Web of Science ID 000244390100008

    View details for PubMedID 17160352

  • Structure and mechanism of the 6-deoxyerythronolide B synthase ANNUAL REVIEW OF BIOCHEMISTRY Khosla, C., Tang, Y., Chen, A. Y., Schnarr, N. A., Cane, D. E. 2007; 76: 195-221

    Abstract

    6-Deoxyerythronolide B, the macrocyclic aglycone of the antibiotic erythromycin, is synthesized by a polyketide synthase (PKS) that has emerged as the prototypical modular megasynthase. A variety of molecular biological, protein chemical, and biosynthetic experiments over the past two decades have yielded insights into its mechanistic features. More recently, high-resolution structural images of portions of the 6-deoxyerythronolide B synthase have provided a platform for interpreting this wealth of biochemical data, while at the same time presenting a fundamentally new basis for the design of more detailed investigations into this remarkable enzyme. For example, the critical roles of domain-domain interactions and nonconserved linkers, as well as large interdomain movements in the structure and function of modular PKSs, have been highlighted. In turn, these insights point the way forward for more sophisticated and efficient biosynthetic engineering of complex polyketide natural products.

    View details for DOI 10.1146/annurev.biochem.76.053105.093515

    View details for Web of Science ID 000249336800010

    View details for PubMedID 17328673

  • Structure-activity relationship analysis of the selective inhibition of transglutaminase 2 by dihydroisoxazoles JOURNAL OF MEDICINAL CHEMISTRY Watts, R. E., Siegel, M., Khosla, C. 2006; 49 (25): 7493-7501

    Abstract

    Human transglutaminase 2 (TG2) is believed to play an important role in the pathogenesis of various human disorders including celiac sprue, certain neurological diseases, and some types of cancer. Selective inhibition of TG2 should therefore enable further investigation of its role in physiology and disease and may lead to effective clinical treatment. Recently we showed that certain 3-halo-4-,5-dihydroisoxazole containing compounds are selective inhibitors of human TG2 with promising pharmacological activities. Here, we present definitive evidence that this class of compounds targets the active site of human TG2. Structure-activity relationship studies have provided insights into the structural prerequisites for selectivity and have led to the discovery of an inhibitor with about 50-fold higher activity than a prototypical dihydroisoxazole inhibitor with good in vivo activity. A method for preparing enantiomerically enriched analogues was also developed. Our studies show that the 5-(S)-dihydroisoxazole is a markedly better inhibitor of human TG2 than its 5-(R) stereoisomer.

    View details for DOI 10.1021/jm060839a

    View details for Web of Science ID 000242625800024

    View details for PubMedID 17149878

  • Structural and functional studies on SCO1815: A beta-ketoacyl-acyl carrier protein reductase from Streptomyces coelicolor A3(2) BIOCHEMISTRY Tang, Y., Lee, H. Y., Tang, Y., Kim, C., Mathews, I., Khosla, C. 2006; 45 (47): 14085-14093

    Abstract

    Aromatic polyketides are medicinally important natural products produced by type II polyketide synthases (PKSs). Some aromatic PKSs are bimodular and include a dedicated initiation module which synthesizes a non-acetate primer unit. Understanding the mechanism of this initiation module is expected to further enhance the potential for regiospecific modification of bacterial aromatic polyketides. A typical initiation module is comprised of a ketosynthase (KS), an acyl carrier protein (ACP), a malonyl-CoA:ACP transacylase (MAT), an acyl-ACP thioesterase, a ketoreductase (KR), a dehydratase (DH), and an enoyl reductase (ER). Thus far, the identities of the ketoreductase, dehydratase, and enoyl reductase remain a mystery because they are not encoded within the PKS biosynthetic gene cluster. Here we report that SCO1815 from Streptomyces coelicolor A3(2), an uncharacterized homologue of a NADPH-dependent ketoreductase, recognizes and reduces the beta-ketoacyl-ACP intermediate from the initiation module of the R1128 PKS. SCO1815 exhibits moderate specificity for both the acyl chain and the thiol donor. The X-ray crystal structure of SCO1815 was determined to 2.0 A. The structure shows that SCO1815 adopts a Rossmann fold and suggests that a conformational change occurs upon cofactor binding. We propose that a positively charged patch formed by three conserved residues is the ACP docking site. Our findings provide new engineering opportunities for incorporating unnatural primer units into novel polyketides and shed light on the biology of yet another cryptic protein in the S. coelicolor genome.

    View details for DOI 10.1021/bi061187v

    View details for Web of Science ID 000242179100016

    View details for PubMedID 17115703

  • Effect of barley endoprotease EP-B2 on gluten digestion in the intact rat JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Gass, J., Vora, H., Bethune, M. T., Gray, G. M., Khosla, C. 2006; 318 (3): 1178-1186

    Abstract

    Celiac Sprue is a multifactorial disease characterized by an intestinal inflammatory response to ingested gluten. Proteolytically resistant gluten peptides from wheat, rye, and barley persist in the intestinal lumen and elicit an immune response in genetically susceptible individuals. Here, we demonstrate the in vivo ability of a gluten-digesting protease ("glutenase") to accelerate the breakdown of a gluten-rich solid meal. The proenzyme form of endoprotease B, isoform 2 from Hordeum vulgare (EP-B2), was orally administered to adult rats with a solid meal containing 1 g of gluten. Gluten digestion in the stomach and small intestine was monitored as a function of enzyme dose and time by high-performance liquid chromatography and mass spectrometry. In the absence of supplementary EP-B2, gluten was solubilized and proteolyzed to a limited extent in the stomach and was hydrolyzed and assimilated mostly in the small intestine. In contrast, EP-B2 was remarkably effective at digesting gluten in the rat stomach in a dose- and time-dependent fashion. At a 1:25 EP-B2/gluten dose, the gastric concentration of the highly immunogenic 33-mer gliadin peptide was reduced by more than 50-fold within 90 min with no overt signs of toxicity. Evaluation of EP-B2 as an adjunct to diet control is therefore warranted in celiac patients.

    View details for DOI 10.1124/jpet.106.104315

    View details for Web of Science ID 000239878900029

    View details for PubMedID 16757540

  • Production of ansamycin polyketide precursors in Escherichia coli JOURNAL OF ANTIBIOTICS Rude, M. A., Khosla, C. 2006; 59 (8): 464-470

    Abstract

    For the heterologous production of ansamycin polyketides such as rifamycin and geldanamycin in Escherichia coli, a number of unusual but important tools must be engineered into the bacterium. Here we demonstrate efficient production of the starter unit 3-amino-5-hydroxybenzoic acid (AHBA) and the methoxymalonyl extender unit in E. coli. Previous work has demonstrated the production of the ansamycin starter unit AHBA in E. coli in low yield. It was shown that the low yield is primarily due to acetylation of AHBA into N-acetyl-AHBA. Three methods for minimizing this side reaction were evaluated. First, a putative N-arylamine-acetyltransferase (NAT) was deleted from the E. coli chromosome, although this did not alter N-acetyl-AHBA production. Next, E. coli grown in media devoid of glucose yielded a nearly equal mixture of AHBA and N-acetyl-AHBA. Lastly, the NAT inhibitor glycyrrhizic acid was shown to further inhibit the acetylation reaction. The entire set of genes for synthesizing the methoxymalonyl extender unit was transferred from the geldanamycin producer Streptomyces hygroscopicus into E. coli. The pathway specific ACP isolated from the resulting recombinant strain was found to predominantly occur as methyoxymalonyl-ACP. Together, these findings set the stage for engineered biosynthesis of ansamycin polyketides in E. coli.

    View details for Web of Science ID 000240519800004

    View details for PubMedID 17080682

  • The 2.7-angstrom crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B synthase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tang, Y., Kim, C., Mathews, I. I., Cane, D. E., Khosla, C. 2006; 103 (30): 11124-11129

    Abstract

    The x-ray crystal structure of a 194-kDa fragment from module 5 of the 6-deoxyerythronolide B synthase has been solved at 2.7 Angstrom resolution. Each subunit of the homodimeric protein contains a full-length ketosynthase (KS) and acyl transferase (AT) domain as well as three flanking "linkers." The linkers are structurally well defined and contribute extensively to intersubunit or interdomain interactions, frequently by means of multiple highly conserved residues. The crystal structure also reveals that the active site residue Cys-199 of the KS domain is separated from the active site residue Ser-642 of the AT domain by approximately 80 Angstrom. This distance is too large to be covered simply by alternative positioning of a statically anchored, fully extended phosphopantetheine arm of the acyl carrier protein domain from module 5. Thus, substantial domain reorganization appears necessary for the acyl carrier protein to interact successively with both the AT and the KS domains of this prototypical polyketide synthase module. The 2.7-Angstrom KS-AT structure is fully consistent with a recently reported lower resolution, 4.5-Angstrom model of fatty acid synthase structure, and emphasizes the close biochemical and structural similarity between polyketide synthase and fatty acid synthase enzymology.

    View details for DOI 10.1073/pnas.0601924103

    View details for Web of Science ID 000239353900006

    View details for PubMedID 16844787

  • Heterologous expression, purification, refolding, and structural-functional characterization of EP-B2, a self-activating barley cysteine endoprotease CHEMISTRY & BIOLOGY Bethune, M. T., Strop, P., Tang, Y., Sollid, L. M., Khosla, C. 2006; 13 (6): 637-647

    Abstract

    We describe the heterologous expression in Escherichia coli of the proenzyme precursor to EP-B2, a cysteine endoprotease from germinating barley seeds. High yields (50 mg/l) of recombinant proEP-B2 were obtained from E. coli inclusion bodies in shake flask cultures following purification and refolding. The zymogen was rapidly autoactivated to its mature form under acidic conditions at a rate independent of proEP-B2 concentration, suggesting a cis mechanism of autoactivation. Mature EP-B2 was stable and active over a wide pH range and efficiently hydrolyzed a recombinant wheat gluten protein, alpha2-gliadin, at sequences with known immunotoxicity in celiac sprue patients. The X-ray crystal structure of mature EP-B2 bound to leupeptin was solved to 2.2 A resolution and provided atomic insights into the observed subsite specificity of the endoprotease. Our findings suggest that orally administered proEP-B2 may be especially well suited for treatment of celiac sprue.

    View details for DOI 10.1016/j.chembiol.2006.04.008

    View details for Web of Science ID 000238723800012

    View details for PubMedID 16793521

  • Investigating nonribosomal peptide and polyketide biosynthesis by direct detection of intermediates on > 70 kDa polypeptides by using Fourier-transform mass spectrometry CHEMBIOCHEM Hicks, L. M., Mazur, M., Miller, L. M., Dorrestein, P. C., Schnarr, N. A., Khosla, C., Kelleher, N. L. 2006; 7 (6): 904-907

    View details for DOI 10.1002/cbic.200500416

    View details for Web of Science ID 000238171400006

    View details for PubMedID 16642537

  • Rational design of combination enzyme therapy for celiac sprue CHEMISTRY & BIOLOGY Siegel, M., Bethune, M. T., Gass, J., Ehren, J., Xia, J., Johannsen, A., Stuge, T. B., Gray, G. M., Lee, P. P., Khosla, C. 2006; 13 (6): 649-658

    Abstract

    Celiac sprue (also known as celiac disease) is an inheritable, gluten-induced enteropathy of the upper small intestine with an estimated prevalence of 0.5%-1% in most parts of the world. The ubiquitous nature of food gluten, coupled with inadequate labeling regulations in most countries, constantly poses a threat of disease exacerbation and relapse for patients. Here, we demonstrate that a two-enzyme cocktail comprised of a glutamine-specific cysteine protease (EP-B2) that functions under gastric conditions and a PEP, which acts in concert with pancreatic proteases under duodenal conditions, is a particularly potent candidate for celiac sprue therapy. At a gluten:EP-B2:PEP weight ratio of 75:3:1, grocery store gluten is fully detoxified within 10 min of simulated duodenal conditions, as judged by chromatographic analysis, biopsy-derived T cell proliferation assays, and a commercial antigluten antibody test.

    View details for DOI 10.1016/j.chembiol.2006.04.009

    View details for Web of Science ID 000238723800013

    View details for PubMedID 16793522

  • Pharmacologic transglutaminase inhibition attenuates drug-primed liver hypertrophy but not Mallory body formation FEBS LETTERS Strnad, P., Siegel, M., Toivola, D. M., Choi, K., Kosek, J. C., Khosla, C., Omary, M. B. 2006; 580 (9): 2351-2357

    Abstract

    Mallory bodies (MBs) are characteristic of several liver disorders, and consist primarily of keratins with transglutaminase-generated keratin crosslinks. We tested the effect of the transglutaminase-2 (TG2) inhibitor KCC009 on MB formation in a mouse model fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). KCC009 decreased DDC-induced liver enlargement without affecting MB formation or extent of liver injury. TG2 protein and activity increased after DDC feeding and localized within and outside hepatocytes. KCC009 inhibited DDC-induced hepatomegaly by affecting hepatocyte cell size rather than proliferation. Hence, TG2 is a potential mediator of injury-induced hepatomegaly via modulation of hepatocyte hypertrophy, and KCC009-mediated TG2 inhibition does not affect mouse MB formation.

    View details for DOI 10.1016/j.febslet.2006.03.051

    View details for Web of Science ID 000237012800032

    View details for PubMedID 16616523

  • Extender unit and acyl carrier protein specificity of ketosynthase domains of the 6-deoxyerythronolide B synthase JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Chen, A. Y., Schnarr, N. A., Kim, C. Y., Cane, D. E., Khosla, C. 2006; 128 (9): 3067-3074

    Abstract

    Polyketide synthases (PKSs) catalyze the production of numerous biologically important natural products via repeated decarboxylative condensation reactions. Modular PKSs, such as the 6-deoxyerythronolide B synthase (DEBS), consist of multiple catalytic modules, each containing a unique set of covalently linked catalytic domains. To better understand the engineering opportunities of these assembly lines, the extender unit and acyl carrier protein (ACP) specificity of keto synthase (KS) domains from modules 3 and 6 of DEBS were analyzed. These studies were undertaken with a newly developed didomain [KS][AT] construct, which lacks its own ACP domain and can therefore be interrogated with homologous or heterologous ACP or acyl-ACP substrates. By substituting the natural methylmalonyl extender unit with a malonyl group, a modest role was demonstrated for the KS in recognition of the nucleophilic substrate. The KS domain from module 3 of DEBS was found to exhibit a distinct ACP-recognition profile from the KS domain of module 6. On the basis of the above kinetic insights, a hybrid module was constructed ([KS3][AT3][KR5][ACP5][TE]) which displayed substrate recognition and elongation capabilities consistent with the natural module 3 protein. Unlike module 3, however, which lacks a ketoreductase (KR) domain, the hybrid module was able to catalyze reduction of the beta-ketothioester product of chain elongation. The high expression level and functionality of this hybrid protein demonstrates the usefulness of kinetic analysis for hybrid module design.

    View details for DOI 10.1021/ja058093d

    View details for Web of Science ID 000235942000060

    View details for PubMedID 16506788

  • Inhibition of HLA-DQ2-mediated antigen presentation by analogues of a high affinity 33-residue peptide from alpha 2-gliadin JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Xia, J., Siegel, M., Bergseng, E., Sollid, L. M., Khosla, C. 2006; 128 (6): 1859-1867

    Abstract

    Human leukocyte antigen DQ2 is a class II major histocompatibility complex protein that plays a critical role in the pathogenesis of Celiac Sprue by binding to epitopes derived from dietary gluten and triggering the inflammatory response of disease-specific T cells. Inhibition of DQ2-mediated antigen presentation in the small intestinal mucosa of Celiac Sprue patients therefore represents a potentially attractive mode of therapy for this widespread but unmet medical need. Starting from a pro-inflammatory, proteolytically resistant, 33-residue peptide, LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF, we embarked upon a systematic effort to dissect the relationships between peptide structure and DQ2 affinity and to translate these insights into prototypical DQ2 blocking agents. Three structural determinants within the first 20 residues of this 33-mer peptide, including a PQPELPYPQ epitope, its N-terminal flanking sequence, and a downstream Glu residue, were found to be important for DQ2 binding. Guided by the X-ray crystal structure of DQ2, the L11 and L18 residues in the truncated 20-mer analogue were replaced with sterically bulky groups so as to retain high DQ2 affinity but abrogate T cell recognition. A dimeric ligand, synthesized by regiospecific coupling of the 20-mer peptide with a bifunctional linker, was identified as an especially potent DQ2 binding agent. Two such ligands were able to attenuate the proliferation of disease-specific T cell lines in response to gluten antigens and, therefore, represent prototypical examples of pharmacologically suitable DQ2 blocking agents for the potential treatment of Celiac Sprue.

    View details for DOI 10.1021/ja056423o

    View details for Web of Science ID 000235452200025

    View details for PubMedID 16464085

  • Macrolactonization to 10-deoxymethynolide catalyzed by the recombinant thioesterase of the picromycin/methymycin polyketide synthase BIOORGANIC & MEDICINAL CHEMISTRY LETTERS He, W. G., Wu, J. Q., Khosla, C., Cane, D. E. 2006; 16 (2): 391-394

    Abstract

    The recombinant thioesterase (TE) domain of the picromycin/methymycin synthase (PICS) catalyzes the macrolactonization of 3, the N-acetylcysteamine thioester of seco-10-deoxymethynolide to generate 10-deoxymethynolide (1) with high efficiency. By contrast, 4, the 7-dihydro derivative of seco-thioester 3, undergoes exclusive hydrolysis by PICS TE to seco-acid 5. The recombinant TE domain of 6-deoxyerythronolide B synthase (DEBS TE) shows the same reaction specificity as PICS TE, but with significantly lower activity.

    View details for DOI 10.1016/j.bmcl.2005.09.077

    View details for Web of Science ID 000233902200031

    View details for PubMedID 16249083

  • Trapping transient protein-protein interactions in polyketide biosynthesis ACS CHEMICAL BIOLOGY Schnarr, N. A., Khosla, C. 2006; 1 (11): 679-680

    Abstract

    Transient biomolecular interactions are essential for biological processes, but strategies for studying them have remained elusive. A paper in this issue shows how natural enzymatic activities can be exploited to examine protein-protein interactions in fatty acid synthase.

    View details for DOI 10.1021/cb600451d

    View details for Web of Science ID 000243895300010

    View details for PubMedID 17184129

  • Modular polyketide synthases: Investigating intermodular communication using 6 deoxyerythronolide B synthase module 2 BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Moffet, D. A., Khosla, C., Cane, D. E. 2006; 16 (1): 213-216

    Abstract

    A novel variant of 6-deoxyerythronolide B synthase (DEBS) module 2 was constructed to explore the balance between protein-protein-mediated intermodular channeling and intrinsic substrate specificity within DEBS. This construct, termed (N3)Mod2+TE, was co-incubated with a complementary, donor form of the same module, (N5)Mod2(C2), as well as with a mutant of (N5)Mod2(C2) with an inactive ketosynthase domain, in order to determine the extent of intermediate channeling versus substrate diffusion into the downstream module.

    View details for DOI 10.1016/j.bmcl.2005.09.017

    View details for Web of Science ID 000233516200045

    View details for PubMedID 16213712

  • Fermentation, purification, formulation, and pharmacological evaluation of a prolyl endopeptidase from Myxococcus xanthus: Implications for Celiac sprue therapy BIOTECHNOLOGY AND BIOENGINEERING Gass, J., Ehren, J., Strohmeier, G., Isaacs, I., Khosla, C. 2005; 92 (6): 674-684

    Abstract

    Celiac Sprue is a multi-factorial disease characterized by an inflammatory response to ingested wheat gluten and similar proteins in rye and barley. Proline-rich gluten peptides from wheat, rye, and barley are relatively resistant to gastrointestinal digestion, and therefore persist in the intestinal lumen to elicit immunopathology in genetically susceptible individuals. In this study, we characterize the in vitro gluten detoxifying properties of a therapeutically promising prolyl endopeptidase from Myxococcus xanthus (MX PEP), and describe the development of a prototypical enteric-coated capsule containing a pharmacologically useful dose of this enzyme. A high-cell density fed-batch fermentation process was developed for overproduction of recombinant MX PEP in E. coli, yielding 0.25-0.4 g/L purified protein. A simple, scalable purification and lyophilization procedure was established that yields >95% pure, highly active and stable enzyme as a dry powder. The dry powder was blended with excipients and encapsulated in a hard gelatin capsule. The resulting capsule was enteric coated using Eudragit L30-D55 polymer coat, which provided sufficient resistance to gastric conditions (> 1 h in 0.01 M HCl, pH 2 with pepsin) and rapid release under duodenal conditions (15-30 min release in pH 6.0 in the presence of trypsin and chymotrypsin). In conjunction with pancreatic enzymes, MX PEP breaks down whole gluten into a product mixture that is virtually indistinguishable from that generated by the Flavobacterium meningosepticum (FM) PEP as judged by chromatographic assays. Competitive studies involving selected immunogenic peptides mixed with whole gluten reveal that both PEPs have a wide range of substrate specificity. Our results support further in vitro and in vivo evaluation of the MX PEP capsule as an oral therapeutic agent for Celiac Sprue patients.

    View details for DOI 10.1002/bit.20643

    View details for Web of Science ID 000233711600002

    View details for PubMedID 16136593

  • Polyketide double bond biosynthesis. Mechanistic analysis of the dehydratase-containing module 2 of the picromycin/methymycin polyketide synthase JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Wu, J. Q., Zaleski, T. J., Valenzano, C., Khosla, C., Cane, D. E. 2005; 127 (49): 17393-17404

    Abstract

    Picromycin/methymycin synthase (PICS) is a modular polyketide synthase (PKS) that is responsible for the biosynthesis of both 10-deoxymethynolide (1) and narbonolide (2), the parent 12- and 14-membered aglycone precursors of the macrolide antibiotics methymycin and picromycin, respectively. PICS module 2 is a dehydratase (DH)-containing module that catalyzes the formation of the unsaturated triketide intermediate using malonyl-CoA as the chain extension substrate. Recombinant PICS module 2+TE, with the PICS thioesterase domain appended to the C-terminus to allow release of polyketide products, was expressed in Escherichia coli. Purified PICS module 2+TE converted malonyl-CoA and 4, the N-acetylcysteamine thioester of (2S,3R)-2-methyl-3-hydroxypentanoic acid, to a 1:2 mixture of the triketide acid (4S,5R)-4-methyl-5-hydroxy-2-heptenoic acid (5) and (3S,4S,5R)-3,5-dihydroxy-4-methyl-n-heptanoic acid-delta-lactone (10) with a combined kcat of 0.6 min(-1). The triketide lactone 10 is formed by thioesterase-catalyzed cyclization of the corresponding d-3-hydroxyacyl-SACP intermediate, a reaction which competes with dehydration catalyzed by the dehydratase domain. PICS module 2+TE showed a strong preference for the syn-diketide-SNAC 4, with a 20-fold greater kcat/K(m) than the anti-(2S,3S)-diketide-SNAC 14, and a 40-fold advantage over the syn-(2R,3S)-diketide-SNAC 13. PICS module 2(DH(0))+TE, with an inactivated DH domain, produced exclusively 10, while three PICS module 2(KR(0))+TE mutants, with inactivated KR domains, produced exclusively or predominantly the unreduced triketide ketolactone, (4S,5R)-3-oxo-4-methyl-5-hydroxy-n-heptanoic acid-delta-lactone (7). These studies establish for the first time the structure and stereochemistry of the intermediates of a polyketide chain elongation cycle catalyzed by a DH-containing module, while confirming the importance of key active site residues in both KR and DH domains.

    View details for DOI 10.1021/ja055672+

    View details for Web of Science ID 000233917500061

    View details for PubMedID 16332089

  • Orthogonal protein interactions in spore pigment producing and antibiotic producing polyketide synthases JOURNAL OF ANTIBIOTICS Lee, T. S., Khosla, C., Tang, Y. 2005; 58 (10): 663-666

    Abstract

    The actinomycetes produce antibiotics as well as spore pigments during their life cycle by using Type II polyketide synthases (PKSs). Each PKS minimally consists of a ketosynthase heterodimer and an acyl carrier protein. The acyl carrier protein has been shown to be interchangeable among different antibiotic producing Type II PKSs. Surprisingly, we have discovered a fundamental incompatibility between the ketosynthases and acyl carrier proteins from antibiotic producing pathways and those from spore pigment pathways. Although antibiotic PKSs can interact with acyl carrier proteins from spore pigment pathways, spore pigment PKSs are unable to recognize acyl carrier proteins from polyketide antibiotic pathways. This observation provides an insight into a critical mechanism by which natural product biosynthetic specificity is exercised by members of this bacterial family.

    View details for Web of Science ID 000232987100008

    View details for PubMedID 16392683

  • Engineered biosynthesis of aklanonic acid analogues JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lee, T. S., Khosla, C., Tang, Y. 2005; 127 (35): 12254-12262

    Abstract

    Aklanonic acid, an anthraquinone natural product, is a common advanced intermediate in the biosynthesis of several antitumor polyketide antibiotics, including doxorubicin and aclacinomycin A. Intensive semisynthetic and biosynthetic efforts have been directed toward developing improved analogues of these clinically important compounds. The primer unit of such polyfunctional aromatic polyketides is an attractive site for introducing novel chemical functionality, and attempts have been made to modify the primer unit by precursor-directed biosynthesis or protein engineering of the polyketide synthase (PKS). We have previously demonstrated the feasibility of engineering bimodular aromatic PKSs capable of synthesizing unnatural hexaketides and octaketides. In this report, we extend this ability by preparing analogues of aklanonic acid, a decaketide, and its methyl ester. For example, by recombining the R1128 initiation module with the dodecaketide-specific pradimicin PKS, the isobutyryl-primed analogue of aklanonic acid (YT296b, 10) and its methyl ester (YT299b, 12) were prepared. In contrast, elongation modules from dodecaketide-specific spore pigment PKSs were unable to interact with the R1128 initiation module. Thus, in addition to revealing a practical route to new anthracycline antibiotics, we also observed a fundamental incompatibility between antibiotic and spore pigment biosynthesis in the actinomycetes bacteria.

    View details for DOI 10.1021/ja051429z

    View details for Web of Science ID 000231637100050

    View details for PubMedID 16131203

  • Analysis of covalently bound polyketide intermediates on 6-deoxyerythronolide B synthase by tandem proteolysis-mass spectrometry BIOCHEMISTRY Schnarr, N. A., Chen, A. Y., Cane, D. E., Khosla, C. 2005; 44 (35): 11836-11842

    Abstract

    Polyketide natural products are biosynthesized via successive chain-elongation events mediated by elaborate protein assemblies. Facile detection of protein-bound intermediates in these systems will increase our understanding of enzyme reactivity and selectivity. We have developed a tandem proteolysis/mass spectrometric method for monitoring substrate loading and elongation in 6-deoxyerythronolide B synthase (DEBS), responsible for production of the macrolide precursor to erythromycin. Information regarding ketosynthase loading and polyketide unit elongation is readily acquired without need for complex protein or small molecule labels. A panel of structurally related substrates is evaluated through competition experiments and kinetic assays using LC-MS to resolve closely related species. Strong stereochemical effects are observed for ketosynthase substrate specificity. Semiquantitative kinetic analyses allow the resolution of the effects of structural and stereochemical changes on the individual ketosynthase-catalyzed steps of acyl-enzyme formation and polyketide chain extension.

    View details for DOI 10.1021/bi0510781

    View details for Web of Science ID 000231710000021

    View details for PubMedID 16128585

  • Identification and analysis of multivalent proteolytically resistant peptides from gluten: Implications for Celiac Sprue JOURNAL OF PROTEOME RESEARCH Shan, L., Qiao, S. W., Arentz-Hansen, H., Molberg, O., Gray, G. M., Sollid, L. M., Khosla, C. 2005; 4 (5): 1732-1741

    Abstract

    Dietary gluten proteins from wheat, rye, and barley are the primary triggers for the immuno-pathogenesis of Celiac Sprue, a widespread immune disease of the small intestine. Recent molecular and structural analyses of representative gluten proteins, most notably alpha- and gamma-gliadin proteins from wheat, have improved our understanding of these pathogenic mechanisms. In particular, based on the properties of a 33-mer peptide, generated from alpha-gliadin under physiological conditions, a link between digestive resistance and inflammatory character of gluten has been proposed. Here, we report three lines of investigation in support of this hypothesis. First, biochemical and immunological analysis of deletion mutants of alpha-2 gliadin confirmed that the DQ2 restricted T cell response to the alpha-2 gliadin are directed toward the epitopes clustered within the 33-mer. Second, proteolytic analysis of a representative gamma-gliadin led to the identification of another multivalent 26-mer peptide that was also resistant to further gastric, pancreatic and intestinal brush border degradation, and was a good substrate of human transglutaminase 2 (TG2). Analogous to the 33-mer, the synthetic 26-mer peptide displayed markedly enhanced T cell antigenicity compared to monovalent control peptides. Finally, in silico analysis of the gluten proteome led to the identification of at least 60 putative peptides that share the common characteristics of the 33-mer and the 26-mer peptides. Together, these results highlight the pivotal role of physiologically generated, proteolytically stable, TG2-reactive, multivalent peptides in the immune response to dietary gluten in Celiac Sprue patients. Prolyl endopeptidase treatment was shown to abolish the antigenicity of both the 33-mer and the 26-mer peptides, and was also predicted to have comparable effects on other proline-rich putatively immunotoxic peptides identified from other polypeptides within the gluten proteome.

    View details for DOI 10.1021/pr050173t

    View details for Web of Science ID 000232579100036

    View details for PubMedID 16212427

  • Tissue transglutaminase 2 inhibition promotes cell death and chemosensitivity in glioblastomas MOLECULAR CANCER THERAPEUTICS Yuan, L., Choi, K. H., Khosla, C., Zheng, X., Higashikubo, R., Chicoine, M. R., Rich, K. M. 2005; 4 (9): 1293-1302

    Abstract

    Tissue transglutaminase 2 belongs to a family of transglutaminase proteins that confers mechanical resistance from proteolysis and stabilizes proteins. Transglutaminase 2 promotes transamidation between glutamine and lysine residues with the formation of covalent linkages between proteins. Transglutaminase 2 also interacts and forms complexes with proteins important in extracellular matrix organization and cellular adhesion. We have identified the novel finding that treatment of glioblastoma cells with transglutaminase 2 inhibitors promotes cell death and enhances sensitivity to chemotherapy. Treatment with either the competitive transglutaminase 2 inhibitor, monodansylcadaverine, or with highly specific small-molecule transglutaminase 2 inhibitors, KCA075 or KCC009, results in induction of apoptosis in glioblastoma cells. Treatment with these transglutaminase 2 inhibitors resulted in markedly decreased levels of the prosurvival protein, phosphorylated Akt, and its downstream targets. These changes promote a proapoptotic profile with altered levels of multiple intracellular proteins that determine cell survival. These changes include decreased levels of the antiapoptotic proteins, survivin, phosphorylated Bad, and phosphorylated glycogen synthetase kinase 3beta (GSK-3beta), and increased levels of the proapoptotic BH3-only protein, Bim. In vivo studies with s.c. murine DBT glioblastoma tumors treated with transglutaminase 2 inhibitors combined with the chemotherapeutic agent, N-N'-bis (2-chloroethyl)-N-nitrosourea (BCNU), decreased tumor size based on weight by 50% compared with those treated with BCNU alone. Groups treated with transglutaminase 2 inhibitors showed an increased incidence of apoptosis determined with deoxynucleotidyl transferase-mediated biotin nick-end labeling staining. These studies identify inhibition of transglutaminase 2 as a potential target to enhance cell death and chemosensitivity in glioblastomas.

    View details for DOI 10.1158/1535-7163.MCT-04-0328

    View details for Web of Science ID 000231923500002

    View details for PubMedID 16170020

  • Biological chemistry: Just add chlorine NATURE Schnarr, N. A., Khosla, C. 2005; 436 (7054): 1094-1095

    View details for DOI 10.1038/4361094a

    View details for Web of Science ID 000231416600026

    View details for PubMedID 16121161

  • Stereochemical assignment of intermediates in the rifamycin biosynthetic pathway by precursor-directed biosynthesis JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Hartung, I. V., Rude, M. A., Schnarr, N. A., Hunziker, D., Khosla, C. 2005; 127 (32): 11202-11203

    Abstract

    Natural and semisynthetic rifamycins are clinically important inhibitors of bacterial DNA-dependent RNA polymerase. Although the polyketide-nonribosomal peptide origin of the naphthalene core of rifamycin B is well established, the absolute and relative configuration of both stereocenters introduced by the first polyketide synthase module is obscured by aromatization of the naphthalene ring. To decode the stereochemistry of the rifamycin polyketide precursor, we synthesized all four diastereomers of the biosynthetic substrate for module 2 of the rifamycin synthetase in the form of their N-acetylcysteamine (SNAC) thioester. Only one diastereomer was turned over in vivo into rifamycin B, thus establishing the absolute and relative configuration of the native biosynthetic intermediates.

    View details for DOI 10.1021/ja051430y

    View details for Web of Science ID 000231227400002

    View details for PubMedID 16089423

  • Effect of pretreatment of food gluten with prolyl endopeptidase on gluten-induced malabsorption in celiac sprue CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Pyle, G. G., Paaso, B., Anderson, B. E., Allen, D. D., Marti, T., Li, Q., Siegel, M., Khosla, C., Gray, G. M. 2005; 3 (7): 687-694

    Abstract

    We sought to determine whether prolyl endopeptidase (PEP) treatment of food gluten would obviate the intestinal dysfunction produced by small amounts of dietary gluten supplement in patients with celiac sprue.Twenty asymptomatic patients with histologically proven celiac sprue completed a randomized, double-blind, cross-over study involving two 14-day stages. Each patient consumed a low dose of a gluten supplement daily (5 g; equivalent to 1 slice of bread) in 1 stage and gluten pretreated with PEP in the other stage. Patients completed a daily symptom questionnaire and a D-xylose urine excretion and a 72-hour quantitative fecal fat were monitored before and after each stage.Despite clinical remission at baseline, 40% of patients had at least 1 abnormal celiac antibody, 20% had an abnormal urine xylose, and 63% had an abnormal fecal fat test result. There was no difference in symptoms as a function of the type of gluten consumed. In response to gluten not treated with PEP, an appreciable proportion of patients developed malabsorption of fat (7 of 17, 41%) or xylose (8 of 14, 57%). When the gluten was pretreated with PEP, fat malabsorption was avoided in 5 of 7 and xylose malabsorption in 4 of 8 of these same patients.A significant proportion of asymptomatic patients with celiac sprue have abnormal celiac antibodies and fat or carbohydrate malabsorption. Pretreatment of gluten with PEP avoided the development of fat or carbohydrate malabsorption in the majority of those patients who developed fat or carbohydrate malabsorption after a 2-week gluten challenge.

    View details for DOI 10.1053/S1542-3565(05)00366-6

    View details for Web of Science ID 000234105600014

    View details for PubMedID 16206502

  • Main chain hydrogen bond interactions in the binding of proline-rich gluten peptides to the Celiac disease-associated HLA-DQ2 molecule JOURNAL OF BIOLOGICAL CHEMISTRY Bergseng, E., Xia, J., Kim, C. Y., Khosla, C., Sollid, L. M. 2005; 280 (23): 21791-21796

    Abstract

    Binding of peptide epitopes to major histocompatibility complex proteins involves multiple hydrogen bond interactions between the peptide main chain and major histocompatibility complex residues. The crystal structure of HLA-DQ2 complexed with the alphaI-gliadin epitope (LQPFPQPELPY) revealed four hydrogen bonds between DQ2 and peptide main chain amides. This is remarkable, given that four of the nine core residues in this peptide are proline residues that cannot engage in amide hydrogen bonding. Preserving main chain hydrogen bond interactions despite the presence of multiple proline residues in gluten peptides is a key element for the HLA-DQ2 association of celiac disease. We have investigated the relative contribution of each main chain hydrogen bond interaction by preparing a series of N-methylated alphaI epitope analogues and measuring their binding affinity and off-rate constants to DQ2. Additionally, we measured the binding of alphaI-gliadin peptide analogues in which norvaline, which contains a backbone amide hydrogen bond donor, was substituted for each proline. Our results demonstrate that hydrogen bonds at P4 and P2 positions are most important for binding, whereas the hydrogen bonds at P9 and P6 make smaller contributions to the overall binding affinity. There is no evidence for a hydrogen bond between DQ2 and the P1 amide nitrogen in peptides without proline at this position. This is a unique feature of DQ2 and is likely a key parameter for preferential binding of proline-rich gluten peptides and development of celiac disease.

    View details for Web of Science ID 000229557900017

    View details for PubMedID 15826953

  • A new route to designer antibiotics SCIENCE Khosla, C., Tang, Y. 2005; 308 (5720): 367-368

    View details for DOI 10.1126/science.1111415

    View details for Web of Science ID 000228492000039

    View details for PubMedID 15831747

  • Chemistry and biology of dihydroisoxazole derivatives: Selective inhibitors of human transglutaminase 2 CHEMISTRY & BIOLOGY Choi, K., Siegel, M., Piper, J. L., Yuan, L., Cho, E., Strnad, P., Omary, B., Rich, K. M., Khosla, C. 2005; 12 (4): 469-475

    Abstract

    3-halo-4,5-dihydroisoxazoles are attractive warheads for the selective inhibition of nucleophilic active sites in biological systems. A series of 3-bromo-4,5-dihydroisoxazole compounds were prepared and tested for their ability to irreversibly inhibit human transglutaminase 2 (TG2), an enzyme that plays an important role in the pathogenesis of diverse disorders including Celiac Sprue and certain types of cancers. Several compounds showed high specificity for human TG2 (k(inh)/K(I) > 2000 min(-1)M(-1)) but essentially no reactivity (k < 1 min(-1)M(-1)) toward physiological thiols such as glutathione. The pharmacokinetic and pharmacodynamic properties of a prototype dihydroisoxazole inhibitor, 1b, were evaluated; in mice the compound showed good oral bioavailability, short serum half-life and efficient TG2 inhibition in small intestinal tissue, and low toxicity. It also showed excellent synergism with N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU, carmustine) against refractory glioblastoma tumors in mice. A fluorescent dihydroisoxazole inhibitor 5 facilitated microscopic visualization of TG2 endocytosis from the extracellular surface of HCT-116 cells. Together, these findings demonstrate the promise of dihydroisoxazole compounds as probes for the biology of TG2 and its role in human disease.

    View details for DOI 10.1016/j.chembiol.2005.02.007

    View details for Web of Science ID 000229065700012

    View details for PubMedID 15850984

  • Equilibrium and kinetic analysis of the unusual binding behavior of a highly immunogenic gluten peptide to HLA-DQ2 BIOCHEMISTRY Xia, J., Sollid, L. M., Khosla, C. 2005; 44 (11): 4442-4449

    Abstract

    HLA-DQ2 predisposes an individual to celiac sprue by presenting peptides from dietary gluten to intestinal CD4(+) T cells. A selectively deamidated multivalent peptide from gluten (LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF; underlined residues correspond to posttranslational Q --> E alterations) is a potent trigger of DQ2 restricted T cell proliferation. Here we report equilibrium and kinetic measurements of interactions between DQ2 and (i) this highly immunogenic multivalent peptide, (ii) its individual constituent epitopes, (iii) its nondeamidated precursor, and (iv) a reference high-affinity ligand of HLA-DQ2 that is not recognized by gluten-responsive T cells from celiac sprue patients. The deamidated 33-mer peptide efficiently exchanges with a preloaded peptide in the DQ2 ligand-binding groove at pH 5.5 as well as pH 7.3, suggesting that the peptide can be presented to T cells comparably well through the endocytic pathway or via direct loading onto extracellular HLA-DQ2. In contrast, the monovalent peptides, and the nondeamidated precursor, as well as the tight-binding reference peptide show a much poorer ability to exchange with a preloaded peptide in the DQ2 binding pocket, especially at pH 7.3, suggesting that endocytosis of these peptides is a prerequisite for T cell presentation. At pH 5.5 and 7.3, dissociation of the deamidated 33-mer peptide from DQ2 is much slower than dissociation of its constituent monovalent epitopes or the nondeamidated precursor but faster than dissociation of the reference high-affinity peptide. Oligomeric states involving multiple copies of the DQ2 heterodimer bound to a single copy of the multivalent 33-mer peptide are not observed. Together, these results suggest that the remarkable antigenicity of the 33-mer gluten peptide is primarily due to its unusually efficient ability to displace existing ligands in the HLA-DQ2 binding pocket, rather than an extremely low rate of dissociation.

    View details for DOI 10.1021/bi047747c

    View details for Web of Science ID 000227736900034

    View details for PubMedID 15766274

  • Structural and mechanistic analysis of two prolyl endopeptidases: Role of interdomain dynamics in catalysis and specificity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shan, L., Mathews, I. I., Khosla, C. 2005; 102 (10): 3599-3604

    Abstract

    Prolyl endopeptidases (PEPs) are a unique class of serine proteases with considerable therapeutic potential for the treatment of celiac sprue. The crystal structures of two didomain PEPs have been solved in alternative configurations, thereby providing insights into the mode of action of these enzymes. The structure of the Sphingomonas capsulata PEP, solved and refined to 1.8-A resolution, revealed an open configuration of the active site. In contrast, the inhibitor-bound PEP from Myxococcus xanthus was crystallized (1.5-A resolution) in a closed form. Comparative analysis of the two structures highlights a critical role for the domain interface in regulating interdomain dynamics and substrate specificity. Structure-based mutagenesis of the M. xanthus PEP confirms an important role for several interfacial residues. A salt bridge between Arg-572 and Asp-196/Glu-197 appears to act as a latch for opening or closing the didomain enzyme, and Arg-572 and Ile-575 may also help secure the incoming peptide substrate to the open form of the enzyme. Arg-618 and Asp-145 are responsible for anchoring the invariant proline residue in the active site of this postproline-cleaving enzyme. A model is proposed for the docking of a representative substrate PQPQLPYPQPQLP in the active site, where the N-terminal substrate residues interact extensively with the catalytic domain, and the C-terminal residues stretch into the propeller domain. Given the promise of the M. xanthus PEP as an oral therapeutic enzyme for treating celiac sprue, our results provide a strong foundation for further optimization of the PEP's clinically useful features.

    View details for DOI 10.1073/pnas.0408286102

    View details for Web of Science ID 000227533100016

    View details for PubMedID 15738423

  • Future therapeutic options for celiac disease NATURE CLINICAL PRACTICE GASTROENTEROLOGY & HEPATOLOGY Sollid, L. M., Khosla, C. 2005; 2 (3): 140-147

    Abstract

    Celiac disease is a disorder of the small intestine caused by an inappropriate immune response to wheat gluten and similar proteins of barley and rye. At present, the only available treatment is a strict gluten-exclusion diet; hence the need for alternative treatments. Recent advances have improved our understanding of the molecular basis for this disorder and there are several attractive targets for new treatments. Oral enzyme supplementation is designed to accelerate gastrointestinal degradation of proline-rich gluten, especially its proteolytically stable antigenic peptides. Complementary strategies aiming to interfere with activation of gluten-reactive T cells include the inhibition of intestinal tissue transglutaminase activity to prevent selective deamidation of gluten peptides, and blocking the binding of gluten peptides to the HLA-DQ2 or HLA-DQ8 molecules. Other possible treatments include cytokine therapy, and selective adhesion molecule inhibitors that interfere with inflammatory reactions, some of which are already showing promise in the clinic for other gastrointestinal diseases.

    View details for Web of Science ID 000229069300009

    View details for PubMedID 16265155

  • Tissue transglutaminase-mediated formation and cleavage of histamine-gliadin complexes: Biological effects and implications for celiac disease JOURNAL OF IMMUNOLOGY Qiao, S. W., Piper, J., Haraldsen, G., Oynebraten, I., Fleckenstein, B., Molberg, O., Khosla, C., Sollid, L. M. 2005; 174 (3): 1657-1663

    Abstract

    Celiac disease is an HLA-DQ2-associated disorder characterized by an intestinal T cell response. The disease-relevant T cells secrete IFN-gamma upon recognition of gluten peptides that have been deamidated in vivo by the enzyme tissue transglutaminase (transglutaminase 2 (TG2)). The celiac intestinal mucosa contains elevated numbers of mast cells, and increased histamine secretion has been reported in celiac patients. This appears paradoxical because histamine typically biases T cell responses in the direction of Th2 instead of the Th1 pattern seen in the celiac lesions. We report that histamine is an excellent substrate for TG2, and it can be efficiently conjugated to gluten peptides through TG2-mediated transamidation. Histamine-peptide conjugates do not exert agonistic effects on histamine receptors, and scavenging of biologically active histamine by gluten peptide conjugation can have physiological implications and may contribute to the mucosal IFN-gamma response in active disease. Interestingly, TG2 is able to hydrolyze the peptide-histamine conjugates when the concentrations of substrates are lowered, thereby releasing deamidated gluten peptides that are stimulatory to T cells.

    View details for Web of Science ID 000226571300065

    View details for PubMedID 15661929

  • Chain elongation, macrolactonization, and hydrolysis of natural and reduced hexaketide substrates by the picromycin/methymycin polyketide synthase ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Wu, J. Q., He, W. G., Khosla, C., Cane, D. E. 2005; 44 (46): 7557-7560

    View details for DOI 10.1002/anie.200502246

    View details for Web of Science ID 000233694800009

    View details for PubMedID 16247819

  • Biochemical analysis of the substrate specificity of the beta-ketoacyl-acyl carrier protein synthase domain of module 2 of the erythromycin polyketide synthase BIOCHEMISTRY Wu, J. Q., Kinoshita, K., Khosla, C., Cane, D. E. 2004; 43 (51): 16301-16310

    Abstract

    The beta-ketoacyl-acyl carrier protein synthase (KS) domain of the modular 6-deoxyerythronolide B synthase (DEBS) catalyzes the fundamental chain building reaction of polyketide biosynthesis. The KS-catalyzed reaction involves two discrete steps consisting of formation of an acyl-enzyme intermediate generated from the incoming acylthioester substrate and an active site cysteine residue, and the conversion of this intermediate to the beta-ketoacyl-acyl carrier protein product by a decarboxylative condensation with a paired methylmalonyl-SACP. We have determined the rate constants for the individual biochemical steps by a combination of protein acylation and transthioesterification experiments. The first-order rate constant (k(2)) for formation of the acyl-enzyme intermediate from [1-(14)C]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SNAC (2) and recombinant DEBS module 2 is 5.8 +/- 2.6 min(-)(1), with a dissociation constant (K(S)) of 3.5 +/- 2.8 mM. The acyl-enzyme adduct was formed at a near-stoichiometric ratio of approximately 0.8:1. Transthioesterification between unlabeled diketide-SNAC 2 and N-[1-(14)C-acetyl]cysteamine gave a k(exch) of 0.15 +/- 0.06 min(-)(1), with a K(m) for HSNAC of 5.7 +/- 4.9 mM and a K(m) for 2 of 5.3 +/- 0.9 mM. Under the conditions that were used, k(exch) was equal to k(-)(2), the first-order rate constant for reversal of the acyl-enzyme-forming reaction. Since the rate of the decarboxylative condensation is much greater that the rate of reversion to the starting material (k(3) > k(-)(2)), formation of the acyl-enzyme adduct is effectively irreversible, thereby establishing that the observed value of the specificity constant (k(cat)/K(m)) is solely a reflection of the intrinsic substrate specificity of the KS-catalyzed acyl-enzyme-forming reaction. These findings were also extended to a panel of diketide- and triketide-SNAC analogues, revealing that some substrate analogues that are not converted to product by DEBS module 2 form dead-end acyl-enzyme intermediates.

    View details for DOI 10.1021/bi048147g

    View details for Web of Science ID 000225937700032

    View details for PubMedID 15610024

  • Reconstituting modular activity from separated domains of 6-deoxyerythronolide B synthase BIOCHEMISTRY Kim, C. Y., Alekseyev, V. Y., Chen, A. Y., Tang, Y. Y., Cane, D. E., Khosla, C. 2004; 43 (44): 13892-13898

    Abstract

    The hallmark of a type I polyketide synthase (PKS), such as the 6-deoxyerythronolide B synthase (DEBS), is the presence of catalytic modules comprised of covalently fused domains acting together to catalyze one round of chain elongation. In addition to an obligate ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP), a module may also include a ketoreductase (KR), dehydratase (DH), and/or enoyl reductase (ER) domain. The size, flexibility, and fixed domain-domain stoichiometry of these PKS modules present challenges for structural, mechanistic, and protein-engineering studies. Here, we have harnessed the power of limited proteolysis and heterologous protein expression to isolate and characterize individual domains of module 3 of DEBS, a 150-kD protein consisting of a KS, an AT, an ACP, and an inactive KR domain. Two interdomain boundaries were identified via limited proteolysis, which led to the production of a 90-kD KS-AT, a 142-kD KS-AT-KR(0), and a 10-kD ACP as structurally stable stand-alone proteins. Each protein was shown to possess the requisite catalytic properties. In the presence of the ACP, both the KS-AT and the KS-AT-KR(0) proteins were able to catalyze chain elongation as well as the intact parent module. Separation of the KS from the ACP enabled direct interrogation of the KS specificity for both the nucleophilic substrate and the partner ACP. Malonyl and methylmalonyl extender units were found to be equivalent substrates for chain elongation. Whereas ACP2 and ACP4 of DEBS could be exchanged for ACP3, ACP6 was a substantially poorer partner for the KS. Remarkably, the newly identified proteolytic sites were conserved in many PKS modules, raising the prospect of developing improved methods for the construction of hybrid PKS modules by engineering domain fusions at these interdomain junctions.

    View details for DOI 10.1021/bi048418n

    View details for Web of Science ID 000224939400002

    View details for PubMedID 15518537

  • Crystal structure of the beta-subunit of Acyl-CoA carboxylase: Structure-based engineering of substrate specificity BIOCHEMISTRY Diacovich, L., Mitchell, D. L., Pham, H., Gago, G., Melgar, M. M., Khosla, C., Gramajo, H., Tsai, S. C. 2004; 43 (44): 14027-14036

    Abstract

    Acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC) catalyze the carboxylation of acetyl- and propionyl-CoA to generate malonyl- and methylmalonyl-CoA, respectively. Understanding the substrate specificity of ACC and PCC will (1) help in the development of novel structure-based inhibitors that are potential therapeutics against obesity, cancer, and infectious disease and (2) facilitate bioengineering to provide novel extender units for polyketide biosynthesis. ACC and PCC in Streptomyces coelicolor are multisubunit complexes. The core catalytic beta-subunits, PccB and AccB, are 360 kDa homohexamers, catalyzing the transcarboxylation between biotin and acyl-CoAs. Apo and substrate-bound crystal structures of PccB hexamers were determined to 2.0-2.8 A. The hexamer assembly forms a ring-shaped complex. The hydrophobic, highly conserved biotin-binding pocket was identified for the first time. Biotin and propionyl-CoA bind perpendicular to each other in the active site, where two oxyanion holes were identified. N1 of biotin is proposed to be the active site base. Structure-based mutagenesis at a single residue of PccB and AccB allowed interconversion of the substrate specificity of ACC and PCC. The di-domain, dimeric interaction is crucial for enzyme catalysis, stability, and substrate specificity; these features are also highly conserved among biotin-dependent carboxyltransferases. Our findings enable bioengineering of the acyl-CoA carboxylase (ACCase) substrate specificity to provide novel extender units for the combinatorial biosynthesis of polyketides.

    View details for DOI 10.1021/bi049065v

    View details for Web of Science ID 000224939400016

    View details for PubMedID 15518551

  • Comparative biochemical analysis of three bacterial prolyl endopeptidases: implications for coeliac sprue BIOCHEMICAL JOURNAL Shan, L., Martin, T., Sollid, L. M., Gray, G. M., Khosla, C. 2004; 383: 311-318

    Abstract

    Prolyl endopeptidases have potential for treating coeliac sprue, a disease of the intestine caused by proteolytically resistant peptides from proline-rich prolamins of wheat, barley and rye. We compared the properties of three similar bacterial prolyl endopeptidases, including the known enzymes from Flavobacterium meningosepticum (FM) and Sphingomonas capsulate (SC) and a novel enzyme from Myxococcus xanthus (MX). These enzymes were interrogated with reference chromogenic substrates, as well as two related gluten peptides (PQPQLPYPQPQLP and LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), believed to play a key role in coeliac sprue pathogenesis. In vitro and in vivo studies were conducted to evaluate the activity, specificity and acid/protease stability of the enzymes. All peptidases were relatively resistant to acid, pancreatic proteases and membrane peptidases of the small intestinal mucosa. Although their activities against reference substrates were similar, the enzymes exhibited substantial differences with respect to chain length and subsite specificity. SC hydrolysed PQPQLPYPQPQLP well, but had negligible activity against LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF. In contrast, the FM and MX peptidases cleaved both substrates, although the FM enzyme acted more rapidly on LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF than MX. Whereas the FM enzyme showed a preference for Pro-Gln bonds, SC cleaved both Pro-Gln and Pro-Tyr bonds with comparable efficiency, and MX had a modest preference for Pro-(Tyr/Phe) sites over Pro-Gln sites. While a more comprehensive understanding of sequence and chain-length specificity may be needed to assess the relative utility of alternative prolyl endopeptidases for treating coeliac sprue, our present work has illustrated the diverse nature of this class of enzymes from the standpoint of proteolysing complex substrates such as gluten.

    View details for DOI 10.1042/BJ20040907

    View details for Web of Science ID 000224794200013

    View details for PubMedID 15245330

  • Effect of prolyl endopeptidase on digestive-resistant gliadin peptides in vivo JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Piper, J. L., Gray, G. M., Khosla, C. 2004; 311 (1): 213-219

    Abstract

    Many gluten peptides elicit proliferative responses from T cells from Celiac Sprue patients, influencing the pathogenesis of this small intestinal disorder. These peptides are Pro- and Gln-rich in character, suggesting that resistance to proteolysis promotes their toxicity. To test this hypothesis, we analyzed the digestive resistance of a panel of alpha- and gamma-gliadin peptides believed to induce toxicity via diverse mechanisms. Most were highly resistant to gastric and pancreatic protease digestion, but they were digested by intestinal brush-border peptidases. In some instances, there was accumulation of relatively long intermediates. Control peptides from gliadin and myoglobin revealed that digestive resistance depended on factors other than size. Prolyl endopeptidase (PEP) supplementation substantially reduced the concentrations of these peptides. To estimate a pharmacologically useful PEP dose, recombinant PEP was coperfused into rat intestine with the highly digestive-resistant 33-mer peptide LQLQPF(PQPQLPY)(3) PQPQPF (PEP: peptide weight ratio 1:50 to 1:5). PEP dosing experiments indicate significant changes in the average residence time. The in vivo benefit of PEP was verified by coperfusion with a mixture of 33-mer and partially proteolyzed gliadin. These data verify and extend our earlier proposal that gliadin peptides, although resistant to proteolysis, can be processed efficiently by PEP supplementation. Indeed, PEP may be able to treat Celiac Sprue by reducing or eliminating such peptides from the intestine.

    View details for DOI 10.1124/jpet.104.068429

    View details for Web of Science ID 000223896100025

    View details for PubMedID 15143130

  • An antibiotic factory caught in action NATURE STRUCTURAL & MOLECULAR BIOLOGY Keatinge-Clay, A., Maltby, D. A., Medzihradszky, K. F., Khosla, C., Stroud, R. M. 2004; 11 (9): 888-893

    Abstract

    The synthesis of aromatic polyketides, such as actinorhodin, tetracycline and doxorubicin, begins with the formation of a polyketide chain. In type II polyketide synthases (PKSs), chains are polymerized by the heterodimeric ketosynthase-chain length factor (KS-CLF). Here we present the 2.0-A structure of the actinorhodin KS-CLF, which shows polyketides being elongated inside an amphipathic tunnel approximately 17 A in length at the heterodimer interface. The structure resolves many of the questions about the roles of KS and CLF. Although CLF regulates chain length, it does not have an active site; KS must catalyze both chain initiation and elongation. We provide evidence that the first cyclization of the polyketide occurs within the KS-CLF tunnel. The mechanistic details of this central PKS polymerase could guide biosynthetic chemists in designing new pharmaceuticals and polymers.

    View details for DOI 10.1038/nsmb808

    View details for Web of Science ID 000223540200024

    View details for PubMedID 15286722

  • Exploring the biosynthetic potential of bimodular aromatic polyketide synthases TETRAHEDRON Tang, Y., Lee, T. S., Lee, H. Y., Khosla, C. 2004; 60 (35): 7659-7671
  • Reconstitution and characterization of a new desosaminyl transferase, EryCIII, from the erythromycin biosynthetic pathway JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lee, H. Y., Chung, H. S., Hang, C., Khosla, C., Walsh, C. T., Kahne, D., Walker, S. 2004; 126 (32): 9924-9925

    Abstract

    EryCIII converts alpha-mycarosyl erythronolide B into erythromycin D using TDP-d-desosamine as the glycosyl donor. We report the heterologous expression, purification, in vitro reconstitution, and preliminary characterization of EryCIII. Coexpression of EryCIII with the GroEL/ES chaperone complex was found to enhance greatly the expression of soluble EryCIII protein. The enzyme was found to be highly active with a kcat greater than 100 min-1. EryCIII was quite selective for the natural nucleotide sugar donor and macrolide acceptor substrates, unlike several other antibiotic glycosyl transferases with broad specificity such as desVII, oleG2, and UrdGT2. Within detectable limits, neither 6-deoxyerythronolide B nor 10-deoxymethynolide were found to be glycosylated by EryCIII. Furthermore, TDP-d-mycaminose, which only differs from TDP-d-desosamine at the C4 position, could not be transferred to alphaMEB. These studies lay the groundwork for detailed structural and mechanistic analysis of an important member of the desosaminyl transferase family of enzymes.

    View details for DOI 10.1021/ja048836f

    View details for Web of Science ID 000223279300025

    View details for PubMedID 15303858

  • Antigen presentation to celiac lesion-derived T cells of a 33-mer gliadin peptide naturally formed by gastrointestinal digestion JOURNAL OF IMMUNOLOGY Qiao, S. W., Bergseng, E., Molberg, O., Xia, J., Fleckenstein, B., Khosla, C., Sollid, L. M. 2004; 173 (3): 1757-1762

    Abstract

    Celiac disease is an HLA-DQ2-associated disorder characterized by intestinal T cell responses to ingested wheat gluten proteins. A peptide fragment of 33 residues (alpha(2)-gliadin 56-88) produced by normal gastrointestinal proteolysis contains six partly overlapping copies of three T cell epitopes and is a remarkably potent T cell stimulator after deamidation by tissue transglutaminase (TG2). This 33-mer is rich in proline residues and adopts the type II polyproline helical conformation in solution. In this study we report that after deamidation, the 33-mer bound with higher affinity to DQ2 compared with other monovalent peptides harboring gliadin epitopes. We found that the TG2-treated 33-mer was presented equally effectively by live and glutaraldehyde-fixed, EBV-transformed B cells. The TG2-treated 33-mer was also effectively presented by glutaraldehyde-fixed dendritic cells, albeit live dendritic cells were the most effective APCs. A strikingly increased T cell stimulatory potency of the 33-mer compared with a 12-mer peptide was also seen with fixed APCs. The 33-mer showed binding maximum to DQ2 at pH 6.3, higher than maxima found for other high affinity DQ2 binders. The 33-mer is thus a potent T cell stimulator that does not require further processing within APC for T cell presentation and that binds to DQ2 with a pH profile that promotes extracellular binding.

    View details for Web of Science ID 000222807100032

    View details for PubMedID 15265905

  • The acyltransferase homologue from the initiation module of the R1128 polyketide synthase is an acyl-ACP thioesterase that edits acetyl primer units BIOCHEMISTRY Tang, Y., Koppisch, A. T., Khosla, C. 2004; 43 (29): 9546-9555

    Abstract

    Type II polyketide synthases (PKSs) synthesize polyfunctional aromatic polyketides through iterative condensations of malonyl extender units. The biosynthesis of most aromatic polyketides is initiated through an acetate unit derived from decarboxylation of malonyl-acyl carrier protein (ACP). Modification of this primer unit represents a powerful method of generating novel polyketides. We have demonstrated that recombination of the initiation module from the R1128 PKS with heterologous elongation modules afforded regioselectively modified polyketides containing alternative primer units. With the exception of the role of the acyltransferase homologue ZhuC, the catalytic cycle of the initiation module has been well explored. ZhuC, along with the ketosynthase III homologue ZhuH and the ACP(p) ZhuG, is essential for the in vivo biosynthesis of aromatic polyketides derived from non-acetate primer units. Here we have studied the role of ZhuC using PKS proteins reconstituted in vitro. We show that the tetracenomycin (tcm) minimal PKS can be directly primed with non-acetate acyl groups. In the presence of approximately 10 microM hexanoyl-ZhuG or approximately 100 microM hexanoyl-CoA, the tcm minimal PKS synthesized hexanoyl-primed analogues of octaketides SEK4 and SEK4b, as well as acetate-primed decaketides SEK15 and SEK15b at comparable levels. Addition of ZhuC abolished synthesis of the acetate-primed decaketides, resulting in exclusive synthesis of the hexanoyl-primed octaketides. In the absence of alternative acyl donors, ZhuC severely retarded the activity of the tcm minimal PKS. The editing capabilities of ZhuC were directly revealed by demonstrating that ZhuC has 100 times greater specificity for acetyl- and propionyl-ACP as compared to hexanoyl- and octanoyl-ACP. Thus, by purging the acetate primer units that otherwise dominate polyketide chain initiation, ZhuC (and presumably its homologues in other PKSs such as the doxorubicin and frenolicin PKSs) allows alternative primer units to be utilized by the elongation module in vivo. The abilities of other alkylacyl primer units to prime the tcm minimal PKS were also investigated in this report.

    View details for DOI 10.1021/bi049157k

    View details for Web of Science ID 000222964900027

    View details for PubMedID 15260498

  • Precursor-directed biosynthesis of epothilone in Escherichia coli JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Boddy, C. N., Hotta, K., Tse, M. L., Watts, R. E., Khosla, C. 2004; 126 (24): 7436-7437

    Abstract

    Engineered biosynthetic pathways provide a powerful method for generating complex molecules. Precursor-directed biosynthesis, which combines chemical synthesis and enzymatic transformations, allows non-native starting materials to be incorporated into biosynthetic pathways. Using this approach, we achieved the production of the anticancer agent epothilone C in Escherichia coli. An E. coli strain was engineered to express the last three modules of the epothilone biosynthetic pathway (epoD-M6, epoE, and epoF) and the substrate required to complement the biosynthetic enzymes was obtained by chemical synthesis. Under high-density cell culture conditions, the E. coli strain processed exogenously fed synthetic substrate into epothilone C at levels comparable to the native host (1 mg/L) and at higher levels than other heterologous hosts. Importantly, this precursor-directed approach will allow chemical modifications to be introduced into the polyketide backbone and may ultimately provide access to epothilone analogues with improved pharmacological properties in quantities sufficient for clinical development.

    View details for DOI 10.1021/ja048108s

    View details for Web of Science ID 000222120900012

    View details for PubMedID 15198579

  • Structural basis for HLA-DQ2-mediated presentation of gluten epitopes in celiac disease PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kim, C. Y., Quarsten, H., Bergseng, E., Khosla, C., Sollid, L. M. 2004; 101 (12): 4175-4179

    Abstract

    Celiac disease, also known as celiac sprue, is a gluten-induced autoimmune-like disorder of the small intestine, which is strongly associated with HLA-DQ2. The structure of DQ2 complexed with an immunogenic epitope from gluten, QLQPFPQPELPY, has been determined to 2.2-A resolution by x-ray crystallography. The glutamate at P6, which is formed by tissue transglutaminase-catalyzed deamidation, is an important anchor residue as it participates in an extensive hydrogen-bonding network involving Lys-beta71 of DQ2. The gluten peptide-DQ2 complex retains critical hydrogen bonds between the MHC and the peptide backbone despite the presence of many proline residues in the peptide that are unable to participate in amide-mediated hydrogen bonds. Positioning of proline residues such that they do not interfere with backbone hydrogen bonding results in a reduction in the number of registers available for gluten peptides to bind to MHC class II molecules and presumably impairs the likelihood of establishing favorable side-chain interactions. The HLA association in celiac disease can be explained by a superior ability of DQ2 to bind the biased repertoire of proline-rich gluten peptides that have survived gastrointestinal digestion and that have been deamidated by tissue transglutaminase. Finally, surface-exposed proline residues in the proteolytically resistant ligand were replaced with functionalized analogs, thereby providing a starting point for the design of orally active agents for blocking gluten-induced toxicity.

    View details for DOI 10.1073/pnas.0306885101

    View details for Web of Science ID 000220472200035

    View details for PubMedID 15020763

  • Engineered biosynthesis of regioselectively modified aromatic polyketides using bimodular polyketide synthases. PLoS biology Tang, Y., Lee, T. S., Khosla, C. 2004; 2 (2): E31-?

    Abstract

    Bacterial aromatic polyketides such as tetracycline and doxorubicin are a medicinally important class of natural products produced as secondary metabolites by actinomyces bacteria. Their backbones are derived from malonyl-CoA units by polyketide synthases (PKSs). The nascent polyketide chain is synthesized by the minimal PKS, a module consisting of four dissociated enzymes. Although the biosynthesis of most aromatic polyketide backbones is initiated through decarboxylation of a malonyl building block (which results in an acetate group), some polyketides, such as the estrogen receptor antagonist R1128, are derived from nonacetate primers. Understanding the mechanism of nonacetate priming can lead to biosynthesis of novel polyketides that have improved pharmacological properties. Recent biochemical analysis has shown that nonacetate priming is the result of stepwise activity of two dissociated PKS modules with orthogonal molecular recognition features. In these PKSs, an initiation module that synthesizes a starter unit is present in addition to the minimal PKS module. Here we describe a general method for the engineered biosynthesis of regioselectively modified aromatic polyketides. When coexpressed with the R1128 initiation module, the actinorhodin minimal PKS produced novel hexaketides with propionyl and isobutyryl primer units. Analogous octaketides could be synthesized by combining the tetracenomycin minimal PKS with the R1128 initiation module. Tailoring enzymes such as ketoreductases and cyclases were able to process the unnatural polyketides efficiently. Based upon these findings, hybrid PKSs were engineered to synthesize new anthraquinone antibiotics with predictable functional group modifications. Our results demonstrate that (i) bimodular aromatic PKSs present a general mechanism for priming aromatic polyketide backbones with nonacetate precursors; (ii) the minimal PKS controls polyketide chain length by counting the number of atoms incorporated into the backbone rather than the number of elongation cycles; and (iii) in contrast, auxiliary PKS enzymes such as ketoreductases, aromatases, and cyclases recognize specific functional groups in the backbone rather than overall chain length. Among the anthracyclines engineered in this study were compounds with (i) more superior activity than R1128 against the breast cancer cell line MCF-7 and (ii) inhibitory activity against glucose-6-phosphate translocase, an attractive target for the treatment of Type II diabetes.

    View details for PubMedID 14966533

  • Engineered biosynthesis of regioselectively modified aromatic polyketides using bimodular polyketide synthases PLOS BIOLOGY Tang, Y., Lee, T. S., Khosla, C. 2004; 2 (2): 227-238
  • Manipulation and analysis of polyketide synthases PROTEIN ENGINEERING Kumar, P., Khosla, C., Tang, Y. 2004; 388: 269-293

    View details for Web of Science ID 000223657700023

    View details for PubMedID 15289078

  • Timeline - Metabolic engineering for drug discovery and development NATURE REVIEWS DRUG DISCOVERY Khosla, C., Keasling, J. D. 2003; 2 (12): 1019-1025

    View details for DOI 10.1038/nrd1256

    View details for Web of Science ID 000186893200019

    View details for PubMedID 14654799

  • Crystal structure of an Acyl-ACP dehydrogenase from the FK520 polyketide Biosynthetic pathway: Insights into extender unit biosynthesis JOURNAL OF MOLECULAR BIOLOGY Watanabe, K., Khosla, C., Stroud, R. M., Tsai, S. C. 2003; 334 (3): 435-444

    Abstract

    Polyketide synthases (PKSs) synthesize the polyketide cores of pharmacologically important natural products such as the immunosuppressants FK520 and FK506. Understanding polyketide biosynthesis at atomic resolution could present new opportunities for chemo-enzymatic synthesis of complex molecules. The crystal structure of FkbI, an enzyme involved in the biosynthesis of the methoxymalonyl extender unit of FK520, was solved to 2.1A with an R(crys) of 24.4%. FkbI has a similar fold to acyl-CoA dehydrogenases. Notwithstanding this similarity, the surface and substrate-binding site of FkbI reveal key differences from other acyl-CoA dehydrogenases, suggesting that FkbI may recognize an acyl-ACP substrate rather than an acyl-CoA substrate. This structural observation coincided the genetic experiment done by Carroll et al. J. Am. Chem. Soc., 124 (2002) 4176. Although an in vitro assay for FkbI remains elusive, the structural basis for the substrate specificity of FkbI is analyzed by a combination of sequence comparison, docking simulations and structural analysis. A biochemical mechanism for the role of FkbI in the biosynthesis of methoxymalonyl-ACP is proposed.

    View details for DOI 10.1016/j.jmb.2003.10.021

    View details for Web of Science ID 000186710600007

    View details for PubMedID 14623185

  • Enhancing the modularity of the modular polyketide synthases: Transacylation in modular polyketide synthases catalyzed by malonyl-CoA : ACP transacylase JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Kumar, P., Koppisch, A. T., Cane, D. E., Khoslaa, C. 2003; 125 (47): 14307-14312

    Abstract

    Selective incorporation of extender units in modular polyketide synthases is primarily controlled by acyl transferase (AT) domains. The AT domains catalyze transacylation of the extender unit from acyl-CoA to the phosphopantetheine arm of an acyl carrier protein (ACP) domain in the same module. New methods that can modulate the extender unit specificity of individual modules with minimal structural or kinetic perturbations in the engineered module are desirable for the efficient biosynthesis of novel natural product analogues. We have demonstrated that transacylation of malonyl groups onto an AT-null form of a mutant modular polyketide synthase by malonyl-CoA:ACP transacylase is an effective strategy for the engineered biosynthesis of site specifically modified polyketides. Using this strategy, 6-deoxyerythronolide B synthase was engineered to exclusively produce 2-desmethyl-6-deoxyerythronolide B. The productivity of the modified system was comparable to that of the wild-type synthase in vitro and in vivo.

    View details for DOI 10.1021/ja037429l

    View details for Web of Science ID 000186722200046

    View details for PubMedID 14624579

  • A switch for the transfer of substrate between nonribosomal peptide and polyketide modules of the rifamycin synthetase assembly line JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Admiraal, S. J., Khosla, C., Walsh, C. T. 2003; 125 (45): 13664-13665

    Abstract

    A nonribosomal peptide synthetase (NRPS) loading module and a polyketide synthase (PKS) elongation module catalyze the preliminary steps in the biosynthesis of the rifamycin antibiotics. A benzoate molecule is covalently attached to the phosphopantetheine arm of the thiolation domain of the loading module when its reaction partner methylmalonyl-CoA is absent. Occupancy of the thiolation domain of the elongation module by a methylmalonyl moiety appears to trigger intermodular transfer of benzoate to the ketosynthase domain of the elongation module. This transthiolation event is fast relative to the initial loading of benzoate onto the loading module. It will be of interest to determine if these results are generally true for intermodular acyl transfer in other NRPS-PKS and PKS assembly lines.

    View details for DOI 10.1021/ja0379060

    View details for Web of Science ID 000186424800021

    View details for PubMedID 14599196

  • Precursor-directed polyketide biosynthesis in Escherichia coli BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Kinoshita, K., Pfeifer, B. A., Khosla, C., Cane, D. E. 2003; 13 (21): 3701-3704

    Abstract

    Precursor-directed polyketide biosynthesis was demonstrated in the heterologous host Escherichia coli. Several diketide and triketide substrates were fed to a recombinant E. coli strain containing a variant form of deoxyerythronolide B synthase (DEBS) from which the first elongation module was deleted resulting in successful macrolactone formation from the diketide, but not the triketide, substrates.

    View details for DOI 10.1016/j.bmcl.2003.08.008

    View details for Web of Science ID 000185992400014

    View details for PubMedID 14552761

  • Biosynthesis of yersiniabactin, a complex polyketide-nonribosomal peptide, using Escherichia coli as a heterologous host APPLIED AND ENVIRONMENTAL MICROBIOLOGY Pfeifer, B. A., Wang, C. C., Walsh, C. T., Khosla, C. 2003; 69 (11): 6698-6702

    Abstract

    The medicinal value associated with complex polyketide and nonribosomal peptide natural products has prompted biosynthetic schemes dependent upon heterologous microbial hosts. Here we report the successful biosynthesis of yersiniabactin (Ybt), a model polyketide-nonribosomal peptide hybrid natural product, using Escherichia coli as a heterologous host. After introducing the biochemical pathway for Ybt into E. coli, biosynthesis was initially monitored qualitatively by mass spectrometry. Next, production of Ybt was quantified in a high-cell-density fermentation environment with titers reaching 67 +/- 21 (mean +/- standard deviation) mg/liter and a volumetric productivity of 1.1 +/- 0.3 mg/liter-h. This success has implications for basic and applied studies on Ybt biosynthesis and also, more generally, for future production of polyketide, nonribosomal peptide, and mixed polyketide-nonribosomal peptide natural products using E. coli.

    View details for DOI 10.1128/AEM.69.11.6698-6702.2003

    View details for Web of Science ID 000186427800046

    View details for PubMedID 14602630

  • Understanding substrate specificity of polyketide synthase modules by generating hybrid multimodular synthases JOURNAL OF BIOLOGICAL CHEMISTRY Watanabe, K., Wang, C. C., Boddy, C. N., Cane, D. E., Khosla, C. 2003; 278 (43): 42020-42026

    Abstract

    Modular polyketide biosynthesis can be harnessed to generate rationally designed complex natural products through bioengineering. A detailed understanding of the features that govern transfer and processing of polyketide biosynthetic intermediates is crucial to successfully engineer new polyketide pathways. Previous studies have shown that substrate stereochemistry and protein-protein interactions between polyketide synthase modules are both important factors in this process. Here we investigated the substrate tolerance of different polyketide modules and assessed the relative importance of inter-module chain transfer versus chain elongation activity of some of these modules. By constructing a variety of hybrid modular polyketide synthase systems and assaying their ability to generate polyketide products, it was determined that the substrate tolerance of each individual ketosynthase domain is an important parameter for the successful recombination of polyketide synthase modules. Surprisingly, however, failure by a module to process a candidate substrate was not due to its inability to bind to it. Rather, it appeared to result from a blockage in carbon-carbon bond formation, suggesting that proper orientation of the initially formed acyl thioester in the ketosynthase active site was important for the enzyme-catalyzed decarboxylative condensation reaction.

    View details for DOI 10.1074/jbc.M305339200

    View details for Web of Science ID 000185989500057

    View details for PubMedID 12923197

  • Polyketide chain length control by chain length factor JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Tang, Y., Tsai, S. C., Khosla, C. 2003; 125 (42): 12708-12709

    Abstract

    Bacterial aromatic polyketides are pharmacologically important natural products. A critical parameter that dictates product structure is the carbon chain length of the polyketide backbone. Systematic manipulation of polyketide chain length represents a major unmet challenge in natural product biosynthesis. Polyketide chain elongation is catalyzed by a heterodimeric ketosynthase. In contrast to homodimeric ketosynthases found in fatty acid synthases, the active site cysteine is absent from the one subunit of this heterodimer. The precise role of this catalytically silent subunit has been debated over the past decade. We demonstrate here that this subunit is the primary determinant of polyketide chain length, thereby validating its designation as chain length factor. Using structure-based mutagenesis, we identified key residues in the chain length factor that could be manipulated to convert an octaketide synthase into a decaketide synthase and vice versa. These results should lead to novel strategies for the engineered biosynthesis of hitherto unidentified polyketide scaffolds.

    View details for DOI 10.1021/ja0378759

    View details for Web of Science ID 000185990300021

    View details for PubMedID 14558809

  • Structure-based mutagenesis of the Malonyl-CoA : Acyl carrier protein transacylase from Streptomyces coelicolor BIOCHEMISTRY Koppisch, A. T., Khosla, C. 2003; 42 (37): 11057-11064

    Abstract

    Malonyl-CoA:acyl carrier protein transacylase (MAT) provides acyl-ACP thioesters for the biosynthesis of aromatic polyketides, and thus is the primary gatekeeper of substrate specificity in type II PKS. A recent report described the X-ray crystal structure of the Streptomyces coelicolor MAT and suggested active site residues which may be important for substrate selectivity [Keatinge-Clay, A. T., et al. (2003) Structure 11, 147-154]. Mutants were made to test the proposed roles of these residues, and the enzymes were characterized kinetically with respect to native and non-native substrates. The activity of the MAT was observed to be greatly attenuated in many of the observed mutants; however, the K(m) for malonyl-CoA was only modestly affected. Our results suggest the MAT uses an active site that is rigorously ordered around the acyl-thioester moiety of the acyl-CoA to facilitate rapid and efficient transacylation to an ACP. Our results also suggest that the MAT does not discriminate against alpha-substituted acyl-CoA thioesters solely on the basis of substrate size.

    View details for DOI 10.1021/bi0349672

    View details for Web of Science ID 000185447100022

    View details for PubMedID 12974642

  • Engineered biosynthesis of an ansamycin polyketide precursor in Escherichia coli PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Watanabe, K., Rude, M. A., Walsh, C. T., Khosla, C. 2003; 100 (17): 9774-9778

    Abstract

    Ansamycins such as rifamycin, ansamitocin, and geldanamycin are an important class of polyketide natural products. Their biosynthetic pathways are especially complex because they involve the formation of 3-amino-5-hydroxybenzoic acid (AHBA) followed by backbone assembly by a hybrid nonribosomal peptide synthetase/polyketide synthase. We have reconstituted the ability to synthesize 2,6-dimethyl-3,5,7-trihydroxy-7-(3'-amino-5'-hydroxyphenyl)-2,4-heptadienoic acid (P8/1-OG), an intermediate in rifamycin biosynthesis, in an extensively manipulated strain of Escherichia coli. The parent strain, BAP1, contains the sfp phosphopantetheinyl transferase gene from Bacillus subtilis, which posttranslationally modifies polyketide synthase and nonribosomal peptide synthetase modules. AHBA biosynthesis in this host required introduction of seven genes from Amycolatopsis mediterranei, which produces rifamycin, and Actinosynnema pretiosum, which produces ansamitocin. Because the four-module RifA protein (530 kDa) from the rifamycin synthetase could not be efficiently produced in an intact form in E. coli, it was genetically split into two bimodular proteins separated by matched linker pairs to facilitate efficient inter-polypeptide transfer of a biosynthetic intermediate. A derivative of BAP1 was engineered that harbors the AHBA biosynthetic operon, the bicistronic RifA construct and the pccB and accA1 genes from Streptomyces coelicolor, which enable methylmalonyl-CoA biosynthesis. Fermentation of this strain of E. coli yielded P8/1-OG, an N-acetyl P8/1-OG analog, and AHBA. In addition to providing a fundamentally new route to shikimate and ansamycin-type compounds, this result enables further genetic manipulation of AHBA-derived polyketide natural products with unprecedented power.

    View details for DOI 10.1073/pnas.1632167100

    View details for Web of Science ID 000184926000028

    View details for PubMedID 12888623

  • A specific role of the Saccharopolyspora erythraea thioesterase II gene in the function of modular polyketide synthases MICROBIOLOGY-SGM Hu, Z. H., Pfeifer, B. A., Chao, E., Murli, S., Kealey, J., Carney, J. R., Ashley, G., Khosla, C., Hutchinson, C. R. 2003; 149: 2213-2225

    Abstract

    Bacterial modular polyketide synthase (PKS) genes are commonly associated with another gene that encodes a thioesterase II (TEII) believed to remove aberrantly loaded substrates from the PKS. Co-expression of the Saccharopolyspora erythraea ery-ORF5 TEII and eryA genes encoding 6-deoxyerythronolide B synthase (DEBS) in Streptomyces hosts eliminated or significantly lowered production of 8,8'-deoxyoleandolide [15-nor-6-deoxyerythronolide B (15-nor-6dEB)], which arises from an acetate instead of a propionate starter unit. Disruption of the TEII gene in an industrial Sac. erythraea strain caused a notable amount of 15-norerythromycins to be produced by utilization of an acetate instead of a propionate starter unit and also resulted in moderately lowered production of erythromycin compared with the amount produced by the parental strain. A similar behaviour of the TEII gene was observed in Escherichia coli strains that produce 6dEB and 15-methyl-6dEB. Direct biochemical analysis showed that the ery-ORF5 TEII enzyme favours hydrolysis of acetyl groups bound to the loading acyl carrier protein domain (ACP(L)) of DEBS. These results point to a clear role of the TEII enzyme, i.e. removal of a specific type of acyl group from the ACP(L) domain of the DEBS1 loading module.

    View details for DOI 10.1099/mic.0.26015-0

    View details for Web of Science ID 000184846300028

    View details for PubMedID 12904561

  • Ketosynthases in the initiation and elongation modules of aromatic polyketide synthases have orthogonal acyl carrier protein specificity BIOCHEMISTRY Tang, Y., Lee, T. S., Kobayashi, S., Khosla, C. 2003; 42 (21): 6588-6595

    Abstract

    Many bacterial aromatic polyketides are synthesized by type II polyketide synthases (PKSs) which minimally consist of a ketosynthase-chain length factor (KS-CLF) heterodimer, an acyl carrier protein (ACP), and a malonyl-CoA:ACP transacylase (MAT). This minimal PKS initiates polyketide biosynthesis by decarboxylation of malonyl-ACP, which is catalyzed by the KS-CLF complex and leads to incorporation of an acetate starter unit. In non-acetate-primed PKSs, such as the frenolicin (fren) PKS and the R1128 PKS, decarboxylative priming is suppressed in favor of chain initiation with alternative acyl groups. Elucidation of these unusual priming pathways could lead to the engineered biosynthesis of polyketides containing novel starter units. Unique to some non-acetate-primed PKSs is a second catalytic module comprised of a dedicated homodimeric KS, an additional ACP, and a MAT. This initiation module is responsible for starter-unit selection and catalysis of the first chain elongation step. To elucidate the protein-protein recognition features of this dissociated multimodular PKS system, we expressed and purified two priming and two elongation KSs, a set of six ACPs from diverse sources, and a MAT. In the presence of the MAT, each ACP was labeled with malonyl-CoA rapidly. In the presence of a KS-CLF and MAT, all ACPs from minimal PKSs supported polyketide synthesis at comparable rates (k(cat) between 0.17 and 0.37 min(-1)), whereas PKS activity was attenuated by at least 50-fold in the presence of an ACP from an initiation module. In contrast, the opposite specificity pattern was observed with priming KSs: while ACPs from initiation modules were good substrates, ACPs from minimal PKSs were significantly poorer substrates. Our results show that KS-CLF and KSIII recognize orthogonal sets of ACPs, and the additional ACP is indispensable for the incorporation of non-acetate primer units. Sequence alignments of the two classes of ACPs identified a tyrosine residue that is unique to priming ACPs. Site-directed mutagenesis of this amino acid in the initiation and elongation module ACPs of the R1128 PKS confirmed the importance of this residue in modulating interactions between KSs and ACPs. Our study provides new biochemical insights into unusual chain initiation mechanisms of bacterial aromatic PKSs.

    View details for DOI 10.1021/bi0341962

    View details for Web of Science ID 000183210900031

    View details for PubMedID 12767243

  • Expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin/methymycin polyketide synthase JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Yin, Y. F., Lu, H. X., Khosla, C., Cane, D. E. 2003; 125 (19): 5671-5676

    Abstract

    Picromycin synthase (PICS) is a multifunctional, modular polyketide synthase (PKS) that catalyzes the conversion of methylmalonyl-CoA to narbonolide and 10-deoxymethynolide, the macrolide aglycone precursors of the antibiotics picromycin and methymycin, respectively. PICS modules 5 and 6 were each expressed in Escherichia coli with a thioesterase domain at the C-terminus to allow release of polyketide products. The substrate specificity of PICS modules 5+TE and 6+TE was investigated using N-acetylcysteamine thioesters of 2-methyl-3-hydroxy-pentanoic acid as diketide analogues of the natural polyketide chain elongation substrates. PICS module 5+TE could catalyze the chain elongation of only the syn diketide (2S,3R)-4, while PICS module 6+TE processed both syn diastereomers, (2S,3R)-4 and (2R,3S)-5, with a 2.5:1 preference in k(cat)/K(m) for 5 but did not turn over either of the two anti diketides. The observed substrate specificity patterns are in contrast to the 15-100:1 preference for 4 over 5 previously established for several modules of the closely related erythromycin PKS, 6-deoxyerythronolide B synthase (DEBS).

    View details for DOI 10.1021/ja034574q

    View details for Web of Science ID 000182769400038

    View details for PubMedID 12733905

  • Mechanistic analysis of acyl transferase domain exchange in polyketide synthase modules JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Hans, M., Hornung, A., Dziarnowski, A., Cane, D. E., Khosla, C. 2003; 125 (18): 5366-5374

    Abstract

    Many polyketides are synthesized by a class of multifunctional enzymes called type I modular polyketide synthases (PKSs). Several reports have described the power of predictively altering polyketide structure by replacing individual PKS domains with homologues from other PKSs. For example, numerous erythromycin analogues have been generated by replacing individual methylmalonyl-specific acyl transferase (AT) domains of the 6-deoxyerythronolide B synthase (DEBS) with malonyl-, ethylmalonyl-, or methoxymalonyl-specific domains. However, the construction of hybrid PKS modules often attenuates product formation both kinetically and distributively. The molecular basis for this mechanistic imperfection is not understood. We have systematically analyzed the impact of replacing an AT domain of DEBS on acyl-AT formation, acyl-CoA:HS-NAc acyl transferase activity, acyl-CoA:ACP acyl transferase activity (nucleophile charging), acyl-SNAc:ketosynthase acyl transferase activity (electrophile charging), and beta-ketoacyl ACP synthase activity (condensation). As usual, domain junctions were located in interdomain regions flanking the AT domain. Kinetic analysis of hybrid modules containing either malonyl transferase or methylmalonyl transferase domains revealed a 15-20-fold decrease in overall turnover numbers of the hybrid modules as compared to the wild-type module. In contrast, both the activity and the specificity of the heterologous AT domains remained unaffected. Moreover, no defects could be detected in the ability of the heterologous AT domains to catalyze acyl-CoA:ACP acyl transfer. Single turnover studies aimed at directly probing the ketosynthase-catalyzed reaction led to two crucial findings. First, wild-type modules catalyzed chain elongation with comparable efficiency regardless of whether methylmalonyl-ACP or malonyl-ACP were the nucleophilic substrates. Second, chain elongation in all hybrid modules tested was seriously attenuated relative to the wild-type module. Our data suggest that, as currently practiced, the most deleterious impact of AT domain swapping is not on the substrate specificity. Rather, it is due to the impaired ability of the KS and ACP domains in the hybrid module to catalyze chain elongation. Consistent with this proposal, limited proteolysis of wild-type and hybrid modules showed major differences in cleavage patterns, especially in the region between the KR and ACP domains.

    View details for DOI 10.1021/ja029539i

    View details for Web of Science ID 000182682700035

    View details for PubMedID 12720450

  • Solution structure and backbone dynamics of the holo form of the frenolicin acyl carrier protein BIOCHEMISTRY Li, Q., Khosla, C., Puglisi, J. D., Liu, C. W. 2003; 42 (16): 4648-4657

    Abstract

    During polyketide biosynthesis, acyl carrier proteins (ACPs) perform the central role of transferring polyketide intermediates between active sites of polyketide synthase. The 4'-phosphopantetheine prosthetic group of a holo-ACP is a long and flexible arm that can reach into different active sites and provide a terminal sulfhydryl group for the attachment of acyl groups through a thioester linkage. We have determined the solution structure and characterized backbone dynamics of the holo form of the frenolicin acyl carrier protein (fren holo-ACP) by nuclear magnetic resonance (NMR). Unambiguous assignments were made for 433 hydrogen atoms, 333 carbon atoms, and 84 nitrogen atoms, representing a total of 94.6% of the assignable atoms in this protein. From 879 meaningful NOEs and 45 angle constraints, a family of 24 structures has been calculated. The solution structure is composed of three major alpha-helices packed in a bundle with three additional short helices in intervening loops; one of the short helices slowly exchanges between two conformations. Superposition of the major helical regions on the mean structure yields average atomic rmsd values of 0.49 +/- 0.09 and 0.91 +/- 0.08 A for backbone and non-hydrogen atoms, respectively. Although the three-helix bundle fold is conserved among acyl carrier proteins involved in fatty acid synthases and polyketide synthases, a detailed comparison revealed that ACPs from polyketide biosynthetic pathways are more related to each other in tertiary fold than to their homologues from fatty acid biosynthetic pathways. Comparison of the free form of ACPs (NMR structures of fren ACP and the Bacillus subtilis ACP) with the substrate-bound form of ACP (crystal structure of butyryl-ACP from Escherichia coli) suggests that conformational exchange plays a role in substrate binding.

    View details for DOI 10.1021/bi0274120

    View details for Web of Science ID 000182460700006

    View details for PubMedID 12705828

  • Intermodular communication in modular polyketide synthases: Structural and mutational analysis of linker mediated protein-protein recognition JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Kumar, P., Li, Q., Cane, D. E., Khosla, C. 2003; 125 (14): 4097-4102

    Abstract

    Modular polyketide synthases (PKSs) present an attractive scaffold for the engineered biosynthesis of novel polyketide products via recombination of naturally occurring enzyme modules with desired catalytic properties. Recent studies have highlighted the pivotal role of short intermodular "linker pairs" in the selective channeling of biosynthetic intermediates between adjacent PKS modules. Using a combination of computer modeling, NMR spectroscopy, cross-linking, and site-directed mutagenesis, we have investigated the mechanism by which a linker pair from the 6-deoxyerythronolide B synthase promotes chain transfer. Our studies support a "coiled-coil" model in which the individual peptides comprising this linker pair adopt helical conformations that associate through a combination of hydrophobic and electrostatic interactions in an antiparallel fashion. Given the important contribution of such linker pair interactions to the kinetics of chain transfer between PKS modules, the ability to rationally modulate linker pair affinity by site-directed mutagenesis could be useful in the construction of optimized hybrid PKSs.

    View details for DOI 10.1021/ja0297537

    View details for Web of Science ID 000182003500035

    View details for PubMedID 12670230

  • Building-block selectivity of polyketide synthases CURRENT OPINION IN CHEMICAL BIOLOGY Liou, G. F., Khosla, C. 2003; 7 (2): 279-284

    Abstract

    For the past decade, polyketide synthases have presented an exciting paradigm for the controlled manipulation of complex natural product structure. These multifunctional enzymes catalyze the biosynthesis of polyketide natural products by stepwise condensation and modification of metabolically derived building blocks. In particular, regioselective modification of polyketide structure is possible by alterations in either intracellular acyl-CoA pools or, more commonly, by manipulation of acyl transferases that act as the primary gatekeepers for building blocks.

    View details for DOI 10.1016/S1367-5931(03)000016-4

    View details for Web of Science ID 000182739700017

    View details for PubMedID 12714062

  • Epothilone C macrolactonization and hydrolysis are catalyzed by the isolated thioesterase domain of epothilone polyketide synthase JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Boddy, C. N., Schneider, T. L., Hotta, K., Walsh, C. T., Khosla, C. 2003; 125 (12): 3428-3429

    Abstract

    Epothilone C is produced by the combined action of one nonribosomal peptide synthetase (NRPS) and nine polyketide synthase (PKS) modules in a multienzyme system. The final step in the biosynthesis is the thioesterase (TE)-catalyzed cyclorelease of epothilone from the EpoF protein. It has been unclear whether isolated PKS TE domains could exhibit macrolactonization activity. Here we demonstrate that the excised epothilone TE domain can catalyze the efficient cyclization of the N-acetylcysteamine thioester of seco-epothilone C to generate epothilone C (kcat/KM = 0.41 +/- 0.03 min-1 mM-1). The TE domain also catalyzes the hydrolysis of both the N-acetylcysteamine thioester of seco-epothilone C (kcat = 0.087 +/- 0.005 min-1, KM = 291 +/- 53 muM) and that of the epothilone C (kcat = 0.67 +/- 0.01 min-1, KM = 117 +/- 5 muM) to form seco-epothilone C.

    View details for DOI 10.1021/ja0298646

    View details for Web of Science ID 000181755800014

    View details for PubMedID 12643694

  • Design, synthesis, and evaluation of gluten peptide analogs as selective inhibitors of human tissue transglutaminase CHEMISTRY & BIOLOGY Hausch, F., Halttunen, T., Maki, M., Khosla, C. 2003; 10 (3): 225-231

    Abstract

    Recent studies have implicated a crucial role for tissue transglutaminase (TG2) in the pathogenesis of Celiac Sprue, a disorder of the small intestine triggered in genetically susceptible individuals by dietary exposure to gluten. Proteolytically stable peptide inhibitors of human TG2 were designed containing acivicin or alternatively 6-diazo-5-oxo-norleucine (DON) as warheads. In biochemical and cell-based assays, the best of these inhibitors, Ac-PQP-(DON)-LPF-NH(2), was considerably more potent and selective than other TG2 inhibitors reported to date. Selective pharmacological inhibition of extracellular TG2 should be useful in exploring the mechanistic implications of TG2-catalyzed modification of dietary gluten, a phenomenon of considerable relevance in Celiac Sprue.

    View details for DOI 10.1016/S1074-5521(03)00045-0

    View details for Web of Science ID 000184092400008

    View details for PubMedID 12670536

  • Catalysis, specificity, and ACP docking site of Streptomyces coelicolor malonyl-CoA : ACP transacylase STRUCTURE Keatinge-Clay, A. T., Shelat, A. A., Savage, D. F., Tsai, S. C., Miercke, L. J., O'Connell, J. D., Khosla, C., Stroud, R. M. 2003; 11 (2): 147-154

    Abstract

    Malonyl-CoA:ACP transacylase (MAT), the fabD gene product of Streptomyces coelicolor A3(2), participates in both fatty acid and polyketide synthesis pathways, transferring malonyl groups that are used as extender units in chain growth from malonyl-CoA to pathway-specific acyl carrier proteins (ACPs). Here, the 2.0 A structure reveals an invariant arginine bound to an acetate that mimics the malonyl carboxylate and helps define the extender unit binding site. Catalysis may only occur when the oxyanion hole is formed through substrate binding, preventing hydrolysis of the acyl-enzyme intermediate. Macromolecular docking simulations with actinorhodin ACP suggest that the majority of the ACP docking surface is formed by a helical flap. These results should help to engineer polyketide synthases (PKSs) that produce novel polyketides.

    View details for Web of Science ID 000180905000006

    View details for PubMedID 12575934

  • Quantitative analysis of loading and extender acyltransferases of modular polyketide synthases BIOCHEMISTRY Liou, G. F., Lau, J., Cane, D. E., Khosla, C. 2003; 42 (1): 200-207

    Abstract

    The acyltransferase (AT) domains of modular polyketide synthases (PKSs) are the primary determinants of building block specificity in polyketide biosynthesis and are therefore attractive targets for protein engineering. Thus far, investigations into the fundamental biochemical properties of AT domains have been hampered by the inability to produce these enzymes as self-standing polypeptides. Here we describe an alternative, generally applicable strategy for overexpression and analysis of AT domains from modular PKSs as truncated didomain proteins (approximately 60 kDa). Recently, we reported the expression and reconstitution of the loading didomain of 6-deoxyerythronolide B synthase (Lau, J., Cane, D. E., and Khosla, C. (2000) Biochemistry 39, 10514-20). By replacing the AT domain of this protein with a methylmalonyl-CoA specific AT domain from module 6 of the 6-deoxyerythronolide B synthase, or alternatively a malonyl-CoA specific AT domain from module 2 of the rapamycin synthase, each of these extender unit AT domains could be overproduced and purified to homogeneity. Using acyl-CoA substrates as acyl group donors and N-acetylcysteamine as the thiol acceptor, we devised a steady-state kinetic assay to probe the properties of these three didomain proteins and selected mutants. Propionyl-CoA was the preferred substrate of the loading didomain, although acetyl- and butyryl-CoA were also accepted with approximately 40-fold-lower specificity. In contrast to the relatively relaxed specificity of the loading AT domain, the methylmalonyl- and malonyl-specific AT domains had high specificity (>1000-fold) toward their natural substrates. The acyl transfer reaction was inhibited by coenzyme A (CoASH) with both a competitive and a noncompetitive component. Use of an exogenous holo-acyl carrier protein (ACP) as an acceptor thiol did not increase the rate of acyl transfer relative to the reaction involving N-acetylcysteamine, suggesting that either the on-rate of the acyl group is rate-limiting or that the apo-ACP component of the didomain protein precludes effective docking of a second ACP onto the AT active site. Mutation of Trp-222 in the loading AT domain to an Arg residue that is universally conserved in all extender unit AT domains failed to enable the loading AT domain to accept methylmalonyl-CoA as an alternative substrate. In contrast, mutation of the equivalent Arg residue in an extender AT domain resulted in a protein with no activity. Together, these results provide a foundation for future structural and mechanistic investigations into the properties of AT domains of modular PKSs.

    View details for DOI 10.1021/bi0268100

    View details for Web of Science ID 000180324300023

    View details for PubMedID 12515555

  • Precursor-directed biosynthesis: Stereospecificity for branched-chain diketides of the beta-ketoacyl-ACP synthase domain 2 of 6-deoxyerythronolide B synthase HELVETICA CHIMICA ACTA Kinoshita, K., Khosla, C., Cane, D. E. 2003; 86 (12): 3889-3907
  • Circular dichroism and nuclear magnetic resonance spectroscopic analysis of immunogenic gluten peptides and their analogs JOURNAL OF BIOLOGICAL CHEMISTRY Parrot, I., Huang, P. C., Khosla, C. 2002; 277 (47): 45572-45578

    Abstract

    Celiac Sprue, or gluten-sensitive enteropathy, is an inheritable human disease of the small intestine that is triggered by the dietary intake of gluten. Recently, several Pro- and Gln-rich peptide sequences (most notably PQPQLPY and analogs) have been identified from gluten with potent immunogenic activity toward CD4(+) T cells from small intestinal biopsies of Celiac Sprue patients. These peptides have three unusual properties. First, they are relatively stable toward further proteolysis by gastric, pancreatic, and intestinal enzymes. Second, they are recognized and deamidated by human tissue transglutaminase (tTGase) with high selectivity. Third, tTGase-catalyzed deamidation enhances their affinity for HLA-DQ2, the disease-specific class II major histocompatibility complex heterodimer. In an attempt to seek a mechanistic explanation for these properties, we undertook secondary structural studies on PQPQLPY and its analogs. Circular dichroism studies on a series of monomeric and dimeric analogs revealed a strong polyproline II helical propensity in a subset of them. Two-dimensional nuclear magnetic resonance spectroscopic analysis confirmed a polyproline II conformation of PQPQLPY, and was also used to elucidate the secondary structure of the most helical variant, (D-P)QPQLPY. Remarkably, a strong correlation was observed between polyproline II content of naturally occurring gluten peptides and the specificity of human tTGase toward these substrates. Analogs with up to two D-amino acid residues retained both polyproline II helical content and transglutaminase affinity. Since the Michaelis constant (K(m)) is the principal determinant of tTGase specificity for naturally occurring gluten peptides and their analogs, our results suggest that the tTGase binding site may have a preference for polyproline II helical substrates. If so, these insights could be exploited for the design of selective small molecule inhibitors of this pharmacologically important enzyme.

    View details for DOI 10.1074/jbc.M207606200

    View details for Web of Science ID 000179404800123

    View details for PubMedID 12324465

  • Crystal structure of the priming beta-ketosynthase from the R1128 polyketide biosynthetic pathway STRUCTURE Pan, H., Tsai, S. C., Meadows, E. S., Miercke, L. J., Keatinge-Clay, A. T., O'Connell, J., Khosla, C., Stroud, R. M. 2002; 10 (11): 1559-1568

    Abstract

    ZhuH is a priming ketosynthase that initiates the elongation of the polyketide chain in the biosynthetic pathway of a type II polyketide, R1128. The crystal structure of ZhuH in complex with the priming substrate acetyl-CoA reveals an extensive loop region at the dimer interface that appears to affect the selectivity for the primer unit. Acetyl-CoA is bound in a 20 A-long channel, which placed the acetyl group against the catalytic triad. Analysis of the primer unit binding site in ZhuH suggests that it can accommodate acyl chains that are two to four carbons long. Selectivity and primer unit size appear to involve the side chains of three residues on the loops close to the dimer interface that constitute the bottom of the substrate binding pocket.

    View details for Web of Science ID 000179175300014

    View details for PubMedID 12429097

  • Insights into channel architecture and substrate specificity from crystal structures of two macrocycle-forming thioesterases of modular polyketide synthases BIOCHEMISTRY Tsai, S. C., Lu, H. X., Cane, D. E., Khosla, C., Stroud, R. M. 2002; 41 (42): 12598-12606

    Abstract

    Modular polyketide synthases (PKSs) synthesize the polyketide cores of pharmacologically important natural products such as erythromycin and picromycin. Understanding PKSs at high resolution could present new opportunities for chemoenzymatic synthesis of complex molecules. The crystal structures of macrocycle-forming thioesterase (TE) domains from the picromycin synthase (PICS) and 6-deoxyerythronolide B synthase (DEBS) were determined to 1.8-3.0 A with an R(crys) of 19.2-24.4%, including three structures of PICS TE (crystallized at pH 7.6, 8.0, and 8.4) and a second crystal form of DEBS TE. As predicted by the previous work on DEBS TE [Tsai, S. C., et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 14808-14813], PICS TE contains an open substrate channel and a hydrophobic dimer interface. Notwithstanding their similarity, the dimer interfaces and substrate channels of DEBS TE and PICS TE reveal key differences. The structural basis for the divergent substrate specificities of DEBS TE and PICS TE is analyzed. The size of the substrate channel increases with increasing pH, presumably due to electrostatic repulsion in the channel at elevated pH. Together, these structures support previous predictions that macrocycle-forming thioesterases from PKSs share the same protein fold, an open substrate channel, a similar catalytic mechanism, and a hydrophobic dimer interface. They also provide a basis for the design of enzymes capable of catalyzing regioselective macrocyclization of natural or synthetic substrates. A series of high-resolution snapshots of a protein channel at different pHs is presented alongside analysis of channel residues, which could help in the redesign of the protein channel architecture.

    View details for DOI 10.1021/bi0260177

    View details for Web of Science ID 000178694000005

    View details for PubMedID 12379102

  • Expression, site-directed mutagenesis, and steady state kinetic analysis of the terminal thioesterase domain of the methymycin/picromycin polyketide synthase BIOCHEMISTRY Lu, H. X., Tsai, S. C., Khosla, C., Cane, D. E. 2002; 41 (42): 12590-12597

    Abstract

    The thioesterase (TE) domain of the methymycin/picromycin synthase (PICS) was functionally expressed in Escherichia coli, and the optimal N-terminal boundary of the recombinant TE was determined. A series of diketide-N-acetylcysteamine (SNAC) thioesters were tested as substrates. PICS TE showed a strong preference for the 2-methyl-3-ketopentanoyl-SNAC substrate 5 over the stereoisomers of the reduced diketides 1-4, with an approximately 1.6:1 preference for the (2R,3S)-2-methyl-3-hydroxy diastereomer 2 over the (2S,3R)-diketide 1. The closely related DEBS TE, the thioesterase from the 6-deoxyerythronolide B synthase, showed a more marked 4.4:1 preference for 2 over 1, with only a slightly greater preference for the 3-ketoacyl-SNAC substrate 5. The roles of several active site residues in PICS TE were examined by site-directed mutagenesis. Serine 148, which is part of the apparent catalytic triad consisting of S148, H268, and D176, was found to be essential for thioesterase activity, while replacement of D176 with asparagine (D176N) gave a mutant thioesterase that retained substantial, albeit reduced, hydrolytic activity toward diketide-SNAC substrates. Mutation of E187 and R191, each of which is thought to play a role in substrate binding, had only minor effects on the relative specificity for diketide substrates 1, 2, and 5. Finally, when PICS TE was fused to the C-terminus of DEBS module 3, the resultant chimeric protein converted diketide 1 with methylmalonyl-CoA to triketide ketolactone 6 with improved catalytic efficiency compared to that of the previously developed DEBS module 3-(DEBS)TE construct.

    View details for DOI 10.1021/bi026006d

    View details for Web of Science ID 000178694000004

    View details for PubMedID 12379101

  • Intestinal digestive resistance of immunodominant gliadin peptides AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY Hausch, F., Shan, L., Santiago, N. A., Gray, G. M., Khosla, C. 2002; 283 (4): G996-G1003

    Abstract

    Two recently identified immunodominant epitopes from alpha-gliadin account for most of the stimulatory activity of dietary gluten on intestinal and peripheral T lymphocytes in patients with celiac sprue. The proteolytic kinetics of peptides containing these epitopes were analyzed in vitro using soluble proteases from bovine and porcine pancreas and brush-border membrane vesicles from adult rat intestine. We showed that these proline-glutamine-rich epitopes are exceptionally resistant to enzymatic processing. Moreover, as estimated from the residual peptide structure and confirmed by exogenous peptidase supplementation, dipeptidyl peptidase IV and dipeptidyl carboxypeptidase I were identified as the rate-limiting enzymes in the digestive breakdown of these peptides. A similar conclusion also emerged from analogous studies with brush-border membrane from a human intestinal biopsy. Supplementation of rat brush-border membrane with trace quantities of a bacterial prolyl endopeptidase led to the rapid destruction of the immunodominant epitopes in these peptides. These results suggest a possible enzyme therapy strategy for celiac sprue, for which the only current therapeutic option is strict exclusion of gluten-containing food.

    View details for DOI 10.1152/ajpgi.00136.2002

    View details for Web of Science ID 000177916600020

    View details for PubMedID 12223360

  • Structural basis for gluten intolerance in Celiac sprue SCIENCE Shan, L., Molberg, O., Parrot, I., Hausch, F., Filiz, F., Gray, G. M., Sollid, L. M., Khosla, C. 2002; 297 (5590): 2275-2279

    Abstract

    Celiac Sprue, a widely prevalent autoimmune disease of the small intestine, is induced in genetically susceptible individuals by exposure to dietary gluten. A 33-mer peptide was identified that has several characteristics suggesting it is the primary initiator of the inflammatory response to gluten in Celiac Sprue patients. In vitro and in vivo studies in rats and humans demonstrated that it is stable toward breakdown by all gastric, pancreatic, and intestinal brush-border membrane proteases. The peptide reacted with tissue transglutaminase, the major autoantigen in Celiac Sprue, with substantially greater selectivity than known natural substrates of this extracellular enzyme. It was a potent inducer of gut-derived human T cell lines from 14 of 14 Celiac Sprue patients. Homologs of this peptide were found in all food grains that are toxic to Celiac Sprue patients but are absent from all nontoxic food grains. The peptide could be detoxified in in vitro and in vivo assays by exposure to a bacterial prolyl endopeptidase, suggesting a strategy for oral peptidase supplement therapy for Celiac Sprue.

    View details for Web of Science ID 000178222000051

    View details for PubMedID 12351792

  • Engineering of molecular and cellular biocatalysts: Selected contributions by James E. Bailey BIOTECHNOLOGY AND BIOENGINEERING Dordick, J. S., Khosla, C. 2002; 79 (5): 490-495

    Abstract

    James (Jay) E. Bailey was a pioneer in biotechnology and biochemical engineering. During his 30 years in academia he made seminal contributions to many fields of chemical engineering science, including catalysis and reaction engineering, bioprocess engineering, mathematical modeling of cellular processes, recombinant DNA technology, enzyme engineering, and metabolic engineering. This article celebrates some of his contributions to the engineering of molecular and cellular biocatalysts, and identifies the influence he had on current and future research in biotechnology.

    View details for DOI 10.1002/bit.10404

    View details for Web of Science ID 000177483600003

    View details for PubMedID 12209820

  • Kinetic and structural analysis of a new group of acyl-CoA carboxylases found in Streptomyces coelicolor A3(2) JOURNAL OF BIOLOGICAL CHEMISTRY Diacovich, L., Peiru, S., Kurth, D., Rodriguez, E., Podesta, F., Khosla, C., Gramajo, H. 2002; 277 (34): 31228-31236

    Abstract

    Two acyl-CoA carboxylases from Streptomyces coelicolor have been successfully reconstituted from their purified components. Both complexes shared the same biotinylated alpha subunit, AccA2. The beta and the epsilon subunits were specific from each of the complexes; thus, for the propionyl-CoA carboxylase complex the beta and epsilon components are PccB and PccE, whereas for the acetyl-CoA carboxylase complex the components are AccB and AccE. The two complexes showed very low activity in the absence of the corresponding epsilon subunits; addition of PccE or AccE dramatically increased the specific activity of the enzymes. The kinetic properties of the two acyl-CoA carboxylases showed a clear difference in their substrate specificity. The acetyl-CoA carboxylase was able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity. The propionyl-CoA carboxylase could not recognize acetyl-CoA as a substrate, whereas the specificity constant for propionyl-CoA was 2-fold higher than for butyryl-CoA. For both enzymes the epsilon subunits were found to specifically interact with their carboxyltransferase component forming a beta-epsilon subcomplex; this appears to facilitate the further interaction of these subunits with the alpha component. The epsilon subunit has been found genetically linked to several carboxyltransferases of different Streptomyces species; we propose that this subunit reflects a distinctive characteristic of a new group of acyl-CoA carboxylases.

    View details for DOI 10.1074/jbc.M203263200

    View details for Web of Science ID 000177579800104

    View details for PubMedID 12048195

  • Process and metabolic strategies for improved production of Escherichia coli-derived 6-deoxyetythronolide B APPLIED AND ENVIRONMENTAL MICROBIOLOGY Pfeifer, B., Hu, Z. H., Licari, P., Khosla, C. 2002; 68 (7): 3287-3292

    Abstract

    Recently, the feasibility of using Escherichia coli for the heterologous biosynthesis of complex polyketides has been demonstrated. In this report, the development of a robust high-cell-density fed-batch procedure for the efficient production of complex polyketides is described. The effects of various physiological conditions on the productivity and titers of 6-deoxyerythronolide B (6dEB; the macrocyclic core of the antibiotic erythromycin) in recombinant cultures of E. coli were studied in shake flask cultures. The resulting data were used as a foundation to develop a high-cell-density fermentation procedure by building upon procedures reported earlier for recombinant protein production in E. coli. The fermentation strategy employed consistently produced approximately 100 mg of 6dEB per liter, whereas shake flask conditions generated between 1 and 10 mg per liter. The utility of an accessory thioesterase (TEII from Saccharopolyspora erythraea) for enhancing the productivity of 6dEB in E. coli was also demonstrated (increasing the final titer of 6dEB to 180 mg per liter). In addition to reinforcing the potential for using E. coli as a heterologous host for wild-type- and engineered-polyketide biosynthesis, the procedures described in this study may be useful for the production of secondary metabolites that are difficult to access by other routes.

    View details for DOI 10.1128/AEM.68.7.3287-3292.2002

    View details for Web of Science ID 000176631600013

    View details for PubMedID 12089005

  • Biochemistry-engineering interface in biochemical engineering AICHE JOURNAL Khosla, C. 2002; 48 (7): 1366-1368
  • Metabolic engineering of a methylmalonyl-CoA mutase-epimerase pathway for complex polyketide biosynthesis in Escherichia coli BIOCHEMISTRY Dayem, L. C., Carney, J. R., Santi, D. V., Pfeifer, B. A., Khosla, C., Kealey, J. T. 2002; 41 (16): 5193-5201

    Abstract

    A barrier to heterologous production of complex polyketides in Escherichia coli is the lack of (2S)-methylmalonyl-CoA, a common extender substrate for the biosynthesis of complex polyketides by modular polyketide synthases. One biosynthetic route to (2S)-methylmalonyl-CoA involves the sequential actions of two enzymes, methylmalonyl-CoA mutase and methylmalonyl-CoA epimerase, which convert succinyl-CoA to (2R)- and then to (2S)-methylmalonyl-CoA. As reported [McKie, N., et al. (1990) Biochem. J. 269, 293-298; Haller, T., et al. (2000) Biochemistry 39, 4622-4629], when genes encoding coenzyme B(12)-dependent methylmalonyl-CoA mutases were expressed in E. coli, the inactive apo-enzyme was produced. However, when cells harboring the mutase genes from Propionibacterium shermanii or E. coli were treated with the B12 precursor hydroxocobalamin, active holo-enzyme was isolated, and (2R)-methylmalonyl-CoA represented approximately 10% of the intracellular CoA pool. When the E. coli BAP1 cell line [Pfeifer, B. A., et al. (2001) Science 291, 1790-1792] harboring plasmids that expressed P. shermanii methylmalonyl-CoA mutase, Streptomyces coelicolor methylmalonyl-CoA epimerase, and the polyketide synthase DEBS (6-deoxyerythronolide B synthase) was fed propionate and hydroxocobalamin, the polyketide 6-deoxyerythronolide B (6-dEB) was produced. Isotopic labeling studies using [(13)C]propionate showed that the starter unit for polyketide synthesis was derived exclusively from exogenous propionate, while the extender units stemmed from methylmalonyl-CoA via the mutase-epimerase pathway. Thus, the introduction of an engineered mutase-epimerase pathway in E. coli enabled the uncoupling of carbon sources used to produce starter and extender units of polyketides.

    View details for DOI 10.1021/bi015593k

    View details for Web of Science ID 000175223400015

    View details for PubMedID 11955068

  • The loading and initial elongation modules of rifamycin synthetase collaborate to produce mixed aryl ketide products-1 BIOCHEMISTRY Admiraal, S. J., Khosla, C., Walsh, C. T. 2002; 41 (16): 5313-5324

    Abstract

    Rifamycin synthetase assembles the chemical backbone that members of the rifamycin family of antibiotics have in common. The synthetase contains a mixed biosynthetic interface between its loading module, which uses a nonribosomal peptide synthetase mechanism, and its initial elongation module, which uses a polyketide synthase mechanism. Biochemical studies of the loading and initial elongation modules of rifamycin synthetase reveal that this bimodular protein (LM-M1) catalyzes the formation of the phenyl ketide 3-hydroxy-2-methyl-3-phenylpropionate via a series of reactions that require benzoate, Mg.ATP, methylmalonyl-CoA, and NADPH. The overall rate of phenyl ketide production appears to be determined by the covalent loading of benzoate onto LM-M1, rather than by subsequent steps such as intermodular transfer of benzoate or condensation of benzoate and methylmalonate. Substituted benzoates that have previously been shown to be substrates for the loading module alone can also be incorporated into the corresponding aryl ketides by LM-M1, suggesting that the bimodular protein has a broad substrate tolerance. Discrimination between the substituted benzoates appears to reside in the benzoate loading reaction, and preincubation of LM-M1 with substituted benzoates and Mg.ATP allows faster downstream reactions to be unmasked. LM-M1 may be a useful biochemical system for exploring interactions between nonribosomal peptide synthetase and polyketide synthase modules.

    View details for DOI 10.1021/bi0200312

    View details for Web of Science ID 000175223400029

    View details for PubMedID 11955082

  • Quantitative analysis of the relative contributions of donor acyl carrier proteins, acceptor ketosynthases, and linker regions to intermodular transfer of intermediates in hybrid polyketide synthases BIOCHEMISTRY Wu, N., Cane, D. E., Khosla, C. 2002; 41 (15): 5056-5066

    Abstract

    6-Deoxyerythronolide B synthase (DEBS) is the modular polyketide synthase (PKS) responsible for the biosynthesis of 6-dEB, the aglycon core of the antibiotic erythromycin. The biosynthesis of 6-dEB proceeds in an assembly-line fashion through the six modules of DEBS, each of which catalyzes a dedicated set of reactions, such that the structure of the final product is determined by the arrangement of modules along the assembly line. This transparent relationship between protein sequence and enzyme function is common to all modular PKSs and makes these enzymes an attractive scaffold for protein engineering through module swapping. One of the fundamental issues relating to module swapping that still needs to be addressed is the mechanism by which intermediates are channeled from one module to the next. While it has been previously shown that short linker regions at the N- and C-termini of adjacent polypeptides play an important role in mediating intermodular transfer, the contributions of other protein-protein interactions have not yet been probed. Here, we investigate the roles of the linker interactions as well as the interactions between the donor acyl carrier protein (ACP) domain and the downstream ketosynthase (KS) domain in various contexts. Linker interactions and ACP-KS interactions make relatively equal contributions at the module 2-module 3 and the module 4-module 5 interfaces in DEBS. In contrast, modules 2 and 6 are more tolerant toward substrates presented by nonnatural ACP domains. This tolerance was exploited for engineering hybrid PKS-PKS and PKS-NRPS (nonribosomal peptide synthetase) junctions and suggests fundamental ground rules for engineering novel chimeric PKSs in the future.

    View details for DOI 10.1021/bi012086u

    View details for Web of Science ID 000175012900035

    View details for PubMedID 11939803

  • High selectivity of human tissue transglutaminase for immunoactive gliadin peptides: Implications for Celiac Sprue BIOCHEMISTRY Piper, J. L., Gray, G. M., Khosla, C. 2002; 41 (1): 386-393

    Abstract

    Celiac Sprue is an HLA DQ2 (or DQ8)-associated autoimmune disorder of the human small intestine that is induced by dietary exposure to wheat gliadin and related proteins from barley, rye, and possibly other food grains. Recently, tissue transglutaminase (tTGase)-catalyzed deamidation of gliadin peptides has been shown to increase their potency for activating patient-derived, gliadin-specific T cells, suggesting that tTGase plays a causative role in the onset of an inflammatory response to toxic food grains. To dissect the molecular recognition features of tTGase for gluten derived peptides, the regioselectivity and steady-state kinetics of tTGase-catalyzed deamidation of known immunogenic peptides were investigated. The specificity of recombinant human tTGase for all immunogenic peptides tested was comparable to and, in some cases, appreciably higher than the specificity for its natural substrate. Although each peptide was glutamine-rich, tTGase exhibited a high degree of regioselectivity for a particular glutamine residue in each peptide. This selectivity correlated well with Q --> E substitutions that have earlier been shown to enhance the immunogenicity of the corresponding gliadin peptides. The specificity of tTGase toward homologues of PQPQLPY, a sequence motif found in immunodominant gliadin peptides, was analyzed in detail. Remarkably, the primary amino acid sequences of wheat-, rye-, and barley-derived proteins included many single-residue variants of this sequence that were high-affinity substrates of tTGase, whereas the closest homologues of this sequence found in rice, corn, or oat proteins were much poorer substrates of tTGase. (Rice, corn, and oats are nontoxic ingredients of the Celiac diet.) No consensus sequence for a high-affinity substrate of tTGase could be derived from our data, suggesting that the secondary structures of these food-grain peptides were important in their recognition by tTGase. Finally, under steady-state turnover conditions, a significant fraction of the tTGase active site was covalently bound to a representative high-affinity immunogenic gliadin peptide, suggesting a common mechanism by which cells responsible for immune surveillance of the intestinal tract recognize and generate an antibody response against both gliadin and tTGase. In addition to providing a quantitative framework for understanding the role of tTGase in Celiac Sprue, our results lay the groundwork for the design of small molecule mimetics of gliadin peptides as selective inhibitors of tTGase.

    View details for DOI 10.1021/bi011715x

    View details for Web of Science ID 000173216500043

    View details for PubMedID 11772038

  • Precursor-directed biosynthesis: Biochemical basis of the remarkable selectivity of the erythromycin polyketide synthase toward unsaturated triketides CHEMISTRY & BIOLOGY Cane, D. E., Kudo, F., Kinoshita, K., Khosla, C. 2002; 9 (1): 131-142

    Abstract

    The structural basis for the striking stereochemical discrimination among triketide analogs has been investigated by incubating a series of N-acetyl cysteamine (-SNAC) esters of unsaturated triketides with DEBS module 2+TE. The triketide analogs were first screened under a standard set of short-term incubation conditions in the presence of the extender substrate methylmalonyl-CoA and NADPH. For those triketide analogs that served as substrates for module 2+TE, the relative specificity, represented by the k(cat)/K(M) values, was quantitated. Triketide diastereomers that were converted in precursor-directed biosynthesis experiments to unsaturated 16-membered ring macrolides by DEBS(KS1(0)) were good to excellent substrates for DEBS module 2+TE, whereas analogs that were converted to the 14-membered ring analogs of 10,11-dehydro-6-deoxyerythronolide B by DEBS(KS1(0)) were not turned over at all by module 2+TE.

    View details for Web of Science ID 000173696100013

    View details for PubMedID 11841945

  • Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase: Versatility from a unique substrate channel PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tsai, S. C., Miercke, L. J., Krucinski, J., Gokhale, R., Chen, J. C., Foster, P. G., Cane, D. E., Khosla, C., Stroud, R. M. 2001; 98 (26): 14808-14813

    Abstract

    As the first structural elucidation of a modular polyketide synthase (PKS) domain, the crystal structure of the macrocycle-forming thioesterase (TE) domain from the 6-deoxyerythronolide B synthase (DEBS) was solved by a combination of multiple isomorphous replacement and multiwavelength anomalous dispersion and refined to an R factor of 24.1% to 2.8-A resolution. Its overall tertiary architecture belongs to the alpha/beta-hydrolase family, with two unusual features unprecedented in this family: a hydrophobic leucine-rich dimer interface and a substrate channel that passes through the entire protein. The active site triad, comprised of Asp-169, His-259, and Ser-142, is located in the middle of the substrate channel, suggesting the passage of the substrate through the protein. Modeling indicates that the active site can accommodate and orient the 6-deoxyerythronolide B precursor uniquely, while at the same time shielding the active site from external water and catalyzing cyclization by macrolactone formation. The geometry and organization of functional groups explain the observed substrate specificity of this TE and offer strategies for engineering macrocycle biosynthesis. Docking of a homology model of the upstream acyl carrier protein (ACP6) against the TE suggests that the 2-fold axis of the TE dimer may also be the axis of symmetry that determines the arrangement of domains in the entire DEBS. Sequence conservation suggests that all TEs from modular polyketide synthases have a similar fold, dimer 2-fold axis, and substrate channel geometry.

    View details for Web of Science ID 000172848800016

    View details for PubMedID 11752428

  • In vitro reconstitution and analysis of the chain initiating enzymes of the R1128 polyketide synthase BIOCHEMISTRY Meadows, E. S., Khosla, C. 2001; 40 (49): 14855-14861

    Abstract

    Biosynthesis of the carbon chain backbone of the R1128 substances is believed to involve the activity of a ketosynthase/chain length factor (ZhuB/ZhuA), an additional ketosynthase (ZhuH), an acyl transferase (ZhuC), and two acyl carrier proteins (ACPs; ZhuG and ZhuN). A subset of these proteins initiate chain synthesis via decarboxylative condensation between an acetyl-, propionyl-, isobutyryl-, or butyryl-CoA derived primer unit and a malonyl-CoA derived extender unit to yield an acetoacetyl-, beta-ketopentanoyl-, 3-oxo-4-methylpentanoyl-, or beta-ketohexanoyl-ACP product, respectively. To investigate the precise roles of ZhuH, ZhuC, ZhuG, and ZhuN, each protein was expressed in Escherichia coli and purified to homogeneity. Although earlier reports had proposed that ZhuC and its homologues played a role in primer unit selection, direct in vitro analysis of ZhuC showed that it was in fact a malonyl-CoA:ACP malonyltransferase (MAT). The enzyme could catalyze malonyl transfer but not acetyl- or propionyl-transfer onto R1128 ACPs or onto ACPs from other biosynthetic pathways, suggesting that ZhuC has broad substrate specificity with respect to the holo-ACP substrate but is specific for malonyl-CoA. Thus, ZhuC supplies extender units to both the initiating and elongating ketosynthases from this pathway. To interrogate the primer unit specificity of ZhuH, the kinetics of beta-ketoacyl-ACP formation in the presence of various acyl-CoAs and malonyl-ZhuG were measured. Propionyl-CoA and isobutyryl-CoA were the two most preferred substrates of ZhuH, although acetyl-CoA and butyryl-CoA could also be accepted and elongated. This specificity is not only consistent with earlier reports demonstrating that R1128B and R1128C are the major products of the R1128 pathway in vivo, but is also in good agreement with the properties of the ZhuH substrate binding pocket, as deduced from a recently solved crystal structure of the enzyme. Finally, to investigate the molecular logic for the occurrence of not one but two ACP genes within the R1128 gene cluster, the inhibition of ZhuH-catalyzed formation of beta-ketopentanoyl-ACP was quantified in the presence of apo-ZhuG or apo-ZhuN. Both apo-proteins were comparable inhibitors of the ZhuH catalyzed reaction, suggesting that the corresponding apo-proteins can be used interchangeably during chain initiation. Together, these results provide direct biochemical insights into the mechanism of chain initiation of an unusual bacterial aromatic PKS.

    View details for Web of Science ID 000172608100015

    View details for PubMedID 11732905

  • Malonyl-CoA : ACP transacylase from Streptomyces coelicolor has two alternative catalytically active nucleophiles BIOCHEMISTRY Dreier, J., Li, Q., Khosla, C. 2001; 40 (41): 12407-12411

    Abstract

    Fatty acids and polyketides are synthesized by mechanistically and evolutionarily related multienzyme systems. Their carbon chain backbones are synthesized via repeated decarboxylative condensations of alpha-carboxylated building blocks onto a growing acyl chain. These alpha-carboxylated building blocks are transferred from the corresponding coenzyme A thioesters onto the phosphopantetheine arm of an acyl carrier protein (ACP) by acyl transferases, which operate by a ping-pong mechanism involving an acyl-O-serine intermediate. In the course of our studies on the malonyl-CoA:ACP transacylase (MAT) from Streptomyces coelicolor, we observed that an active-site Ser (97) --> Ala mutant retains activity as well as the ability to be covalently labeled by (14)C malonyl-CoA. Here we demonstrate that an alternative, catalytically competent nucleophile exists in the active site of this enzyme. Next to the active-site serine is a histidine residue that is conserved in some, but not all acyl transferases. The H96A mutant is also active and can be labeled, but an H96A/S97A double mutant is inactive and cannot be labeled. The ability of H96 to form a malonyl-imidazole adduct was confirmed by proteolysis, followed by radio-HPLC and mass spectrometric analysis of the S97A mutant enzyme. Kinetic analysis revealed that the k(cat) of the S97A mutant was within 10-fold that of the wild-type enzyme, whereas the K(M)s of the two enzymes were comparable. Sequence comparison with the E. coli MAT (whose X-ray structure is known) led to the identification of H201 as the putative base in the serine-histidine catalytic dyad of the S. coelicolor enzyme. The absence of MAT activity in the H201A mutant and the detection of weak activity in the H201Q mutant was consistent with this proposal. The implications of this unexpected finding are discussed.

    View details for Web of Science ID 000171601700021

    View details for PubMedID 11591161

  • Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chiu, H. T., Hubbard, B. K., Shah, A. N., Eide, J., Fredenburg, R. A., Walsh, C. T., Khosla, C. 2001; 98 (15): 8548-8553

    Abstract

    Streptomyces lavendulae produces complestatin, a cyclic peptide natural product that antagonizes pharmacologically relevant protein-protein interactions including formation of the C4b,2b complex in the complement cascade and gp120-CD4 binding in the HIV life cycle. Complestatin, a member of the vancomycin group of natural products, consists of an alpha-ketoacyl hexapeptide backbone modified by oxidative phenolic couplings and halogenations. The entire complestatin biosynthetic and regulatory gene cluster spanning ca. 50 kb was cloned and sequenced. It consisted of 16 ORFs, encoding proteins homologous to nonribosomal peptide synthetases, cytochrome P450-related oxidases, ferredoxins, nonheme halogenases, four enzymes involved in 4-hydroxyphenylglycine (Hpg) biosynthesis, transcriptional regulators, and ABC transporters. The nonribosomal peptide synthetase consisted of a priming module, six extending modules, and a terminal thioesterase; their arrangement and domain content was entirely consistent with functions required for the biosynthesis of a heptapeptide or alpha-ketoacyl hexapeptide backbone. Two oxidase genes were proposed to be responsible for the construction of the unique aryl-ether-aryl-aryl linkage on the linear heptapeptide intermediate. Hpg, 3,5-dichloro-Hpg, and 3,5-dichloro-hydroxybenzoylformate are unusual building blocks that repesent five of the seven requisite monomers in the complestatin peptide. Heterologous expression and biochemical analysis of 4-hydroxyphenylglycine transaminon confirmed its role as an aminotransferase responsible for formation of all three precursors. The close similarity but functional divergence between complestatin and chloroeremomycin biosynthetic genes also presents a unique opportunity for the construction of hybrid vancomycin-type antibiotics.

    View details for Web of Science ID 000169967000063

    View details for PubMedID 11447274

  • Assessing the balance between protein-protein interactions and enzyme-substrate interactions in the channeling of intermediates between polyketide synthase modules JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Wu, N., Tsuji, S. Y., Cane, D. E., Khosla, C. 2001; 123 (27): 6465-6474

    Abstract

    6-Deoxyerythronolide B synthase (DEBS) is the modular polyketide synthase (PKS) that catalyzes the biosynthesis of 6-deoxyerythronolide B (6-dEB), the aglycon precursor of the antibiotic erythromycin. The biosynthesis of 6-dEB exemplifies the extraordinary substrate- and stereo-selectivity of this family of multifunctional enzymes. Paradoxically, DEBS has been shown to be an attractive scaffold for combinatorial biosynthesis, indicating that its constituent modules are also very tolerant of unnatural substrates. By interrogating individual modules of DEBS with a panel of diketides activated as N-acetylcysteamine (NAC) thioesters, it was recently shown that individual modules have a marked ability to discriminate among certain diastereomeric diketides. However, since free NAC thioesters were used as substrates in these studies, the modules were primed by a diffusive process, which precluded involvement of the covalent, substrate-channeling mechanism by which enzyme-bound intermediates are directly transferred from one module to the next in a multimodular PKS. Recent evidence pointing to a pivotal role for protein-protein interactions in the substrate-channeling mechanism has prompted us to develop novel assays to reassess the steady-state kinetic parameters of individual DEBS modules when primed in a more "natural" channeling mode by the same panel of diketide substrates used earlier. Here we describe these assays and use them to quantify the kinetic benefit of linker-mediated substrate channeling in a modular PKS. This benefit can be substantial, especially for intrinsically poor substrates. Examples are presented where the k(cat) of a module for a given diketide substrate increases >100-fold when the substrate is presented to the module in a channeling mode as opposed to a diffusive mode. However, the substrate specificity profiles for individual modules are conserved regardless of the mode of presentation. By highlighting how substrate channeling can allow PKS modules to effectively accept and process intrinsically poor substrates, these studies provide a rational basis for examining the enormous untapped potential for combinatorial biosynthesis via module rearrangement.

    View details for DOI 10.1021/ja010219t

    View details for Web of Science ID 000169835300001

    View details for PubMedID 11439032

  • Enhancing the atom economy of polyketide biosynthetic processes through metabolic engineering BIOTECHNOLOGY PROGRESS Lombo, F., Pfeifer, B., Leaf, T., Ou, S., Kim, Y. S., Cane, D. E., Licari, P., Khosla, C. 2001; 17 (4): 612-617

    Abstract

    Polyketides, a large family of bioactive natural products, are synthesized from building blocks derived from alpha-carboxylated Coenzyme A thioesters such as malonyl-CoA and (2S)-methylmalonyl-CoA. The productivity of polyketide fermentation processes in natural and heterologous hosts is frequently limited by the availability of these precursors in vivo. We describe a metabolic engineering strategy to enhance both the yield and volumetric productivity of polyketide biosynthesis. The genes matB and matC from Rhizobium trifolii encode a malonyl-CoA synthetase and a putative dicarboxylate transport protein, respectively. These proteins can directly convert exogenous malonate and methylmalonate into their corresponding CoA thioesters with an ATP requirement of 2 mol per mol of acyl-CoA produced. Heterologous expression of matBC in a recombinant strain of Streptomyces coelicolor that produces the macrolactone 6-deoxyerythronolide B results in a 300% enhancement of macrolactone titers. The unusual efficiency of the bioconversion is illustrated by the fact that approximately one-third of the methylmalonate units added to the fermentation medium are converted into macrolactones. The direct conversion of inexpensive feedstocks such as malonate and methylmalonate into polyketides represents the most carbon- and energy-efficient route to these high value natural products and has implications for cost-effective fermentation of numerous commercial and development-stage small molecules.

    View details for Web of Science ID 000170338000004

    View details for PubMedID 11485419

  • Remarkably broad substrate tolerance of Malonyl-CoA synthetase, an enzyme capable of intracellular synthesis of polyketide precursors JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Pohl, N. L., Hans, M., Lee, H. Y., Kim, Y. S., Cane, D. E., Khosla, C. 2001; 123 (24): 5822-5823

    View details for Web of Science ID 000169338400035

    View details for PubMedID 11403625

  • Erythromycin biosynthesis. The 4-pro-S hydride of NADPH is utilized for ketoreduction by both module 5 and module 6 of the 6-deoxyerythronolide B synthase BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Yin, Y. F., Gokhale, R., Khosla, C., Cane, D. E. 2001; 11 (12): 1477-1479

    Abstract

    Incubation of chirally deuterated NADPH with 6-deoxyerythronolide B synthase (DEBS) modules 5 and module 6 and analysis of the derived triketide lactones established that the two ketoreductase domains, KR5 and KR6, are both specific for the 4-pro-S hydride of the nicotinamide cofactor.

    View details for Web of Science ID 000169365500001

    View details for PubMedID 11412964

  • The loading module of rifamycin synthetase is an adenylation-thiolation didomain with substrate tolerance for substituted benzoates BIOCHEMISTRY Admiraal, S. J., Walsh, C. T., Khosla, C. 2001; 40 (20): 6116-6123

    Abstract

    The rifamycin synthetase is primed with a 3-amino-5-hydroxybenzoate starter unit by a loading module that contains domains homologous to the adenylation and thiolation domains of nonribosomal peptide synthetases. Adenylation and thiolation activities of the loading module were reconstituted in vitro and shown to be independent of coenzyme A, countering literature proposals that the loading module is a coenzyme A ligase. Kinetic parameters for covalent arylation of the loading module were measured directly for the unnatural substrates benzoate and 3-hydroxybenzoate. This analysis was extended through competition experiments to determine the relative rates of incorporation of a series of substituted benzoates. Our results show that the loading module can accept a variety of substituted benzoates, although it exhibits a preference for the 3-, 5-, and 3,5-disubstituted benzoates that most closely resemble its biological substrate. The considerable substrate tolerance of the loading module of rifamycin synthetase suggests that the module has potential as a tool for generating substituted derivatives of natural products.

    View details for Web of Science ID 000168932900031

    View details for PubMedID 11352749

  • Intellectual border: Two-way traffic CHEMICAL & ENGINEERING NEWS Khosla, C. 2001; 79 (13): 149-149
  • Biosynthesis of complex polyketides in a metabolically engineered strain of E-coli SCIENCE Pfeifer, B. A., Admiraal, S. J., Gramajo, H., Cane, D. E., Khosla, C. 2001; 291 (5509): 1790-1792

    Abstract

    The macrocyclic core of the antibiotic erythromycin, 6-deoxyerythronolide B (6dEB), is a complex natural product synthesized by the soil bacterium Saccharopolyspora erythraea through the action of a multifunctional polyketide synthase (PKS). The engineering potential of modular PKSs is hampered by the limited capabilities for molecular biological manipulation of organisms (principally actinomycetes) in which complex polyketides have thus far been produced. To address this problem, a derivative of Escherichia coli has been genetically engineered. The resulting cellular catalyst converts exogenous propionate into 6dEB with a specific productivity that compares well with a high-producing mutant of S. erythraea that has been incrementally enhanced over decades for the industrial production of erythromycin.

    View details for Web of Science ID 000167320600060

    View details for PubMedID 11230695

  • Biosynthesis of polyketides in heterologous hosts MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS Pfeifer, B. A., Khosla, C. 2001; 65 (1): 106-?

    Abstract

    Polyketide natural products show great promise as medicinal agents. Typically the products of microbial secondary biosynthesis, polyketides are synthesized by an evolutionarily related but architecturally diverse family of multifunctional enzymes called polyketide synthases. A principal limitation for fundamental biochemical studies of these modular megasynthases, as well as for their applications in biotechnology, is the challenge associated with manipulating the natural microorganism that produces a polyketide of interest. To ameliorate this limitation, over the past decade several genetically amenable microbes have been developed as heterologous hosts for polyketide biosynthesis. Here we review the state of the art as well as the difficulties associated with heterologous polyketide production. In particular, we focus on two model hosts, Streptomyces coelicolor and Escherichia coli. Future directions for this relatively new but growing technological opportunity are also discussed.

    View details for Web of Science ID 000167343200004

    View details for PubMedID 11238987

  • Intermodular communication in polyketide syntheses: Comparing the role of protein-protein interactions to those in other multidomain proteins BIOCHEMISTRY Tsuji, S. Y., Wu, N., Khosla, C. 2001; 40 (8): 2317-2325

    Abstract

    Although the role of protein-protein interactions in transducing signals within biological systems has been extensively explored, their relevance to the channeling of intermediates in metabolism is not widely appreciated. Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) are two related families of modular megasynthases that channel covalently bound intermediates from one active site to the next. Recent biochemical studies have highlighted the importance of protein-protein interactions in these chain transfer processes. The information available on this subject is reviewed, and its possible mechanistic implications are placed in context by comparisons with selected well-studied multicomponent protein systems.

    View details for DOI 10.1021/bi002462v

    View details for Web of Science ID 000167121500001

    View details for PubMedID 11327851

  • Selective protein-protein interactions direct channeling of intermediates between polyketide synthase modules BIOCHEMISTRY Tsuji, S. Y., Cane, D. E., Khosla, C. 2001; 40 (8): 2326-2331

    Abstract

    Polyketide synthases (PKSs) have represented fertile targets for rational manipulation via protein engineering ever since their modular architecture was first recognized. However, the mechanistic principles by which biosynthetic intermediates are sequentially channeled between modules remain poorly understood. Here we demonstrate the importance of complementarity in a remarkably simple, repetitive structural motif within these megasynthases that has been implicated to affect intermodular chain transfer [Gokhale, R. S., et al. (1999) Science 284, 482]. The C- and N-terminal ends of adjacent PKS polypeptides are capped by short peptides of 20-40 residues. Mismatched sequences abolish intermodular chain transfer without affecting the activity of individual modules, whereas matched sequences can facilitate the channeling of intermediates between ordinarily nonconsecutive modules. Thus, in addition to substrate-PKS interactions and domain-domain interactions, these short interpolypeptide sequences represent a third determinant of selective chain transfer that must be taken into consideration in the protein engineering of PKSs. Preliminary biophysical studies on synthetic peptide mimics of these linkers suggest that they may adopt coiled-coil conformations.

    View details for Web of Science ID 000167121500002

    View details for PubMedID 11327852

  • Structure-activity relationships within a family of selectively cytotoxic macrolide natural products ORGANIC LETTERS Salomon, A. R., Zhang, Y. B., Seto, H., Khosla, C. 2001; 3 (1): 57-59

    Abstract

    [figure: see text] We describe a semi-synthetic deglycosylated derivative of apoptolidin that retains considerable activity against the mitochondrial ATPase but has greatly reduced cellular cytotoxicity. We also demonstrate that a related antifungal natural product, cytovaricin, inhibits the same molecular target. Structural comparison of these macrolides provides insights into their conserved features that are presumably important for biological activity and identifies promising avenues at the interface of organic synthesis and biosynthesis for the generation of new selective cytotoxic agents.

    View details for DOI 10.1021/ol006767d

    View details for Web of Science ID 000166360000016

    View details for PubMedID 11429871

  • Modular enzymes NATURE Khosla, C., Harbury, P. B. 2001; 409 (6817): 247-252

    Abstract

    Although modular macromolecular devices are encountered frequently in a variety of biological situations, their occurrence in biocatalysis has not been widely appreciated. Three general classes of modular biocatalysts can be identified: enzymes in which catalysis and substrate specificity are separable, multisubstrate enzymes in which binding sites for individual substrates are modular, and multienzyme systems that can catalyse programmable metabolic pathways. In the postgenomic era, the discovery of such systems can be expected to have a significant impact on the role of enzymes in synthetic and process chemistry.

    View details for Web of Science ID 000166316200056

    View details for PubMedID 11196653

  • Apoptolidin, a selective cytotoxic agent, is an inhibitor of F0F1-ATPase CHEMISTRY & BIOLOGY Salomon, A. R., Voehringer, D. W., Herzenberg, L. A., Khosla, C. 2001; 8 (1): 71-80

    Abstract

    Apoptolidin is a macrolide originally identified on the basis of its ability to selectively kill E1A and E1A/E1B19K transformed rat glial cells while not killing untransformed glial cells. The goal of this study was to identify the molecular target of this newly discovered natural product.Our approach to uncovering the mechanism of action of apoptolidin utilized a combination of molecular and cell-based pharmacological assays as well as structural comparisons between apoptolidin and other macrocyclic polyketides with known mechanism of action. Cell killing induced by apoptolidin was independent of p53 status, inhibited by BCL-2, and dependent on the action of caspase-9. PARP was completely cleaved in the presence of 1 microM apoptolidin within 6 h in a mouse lymphoma cell line. Together these results suggested that apoptolidin might target a mitochondrial protein. Structural comparisons between apoptolidin and other macrolides revealed significant similarity between the apoptolidin aglycone and oligomycin, a known inhibitor of mitochondrial F0F1-ATP synthase. The relevance of this similarity was established by demonstrating that apoptolidin is a potent inhibitor of the F0F1-ATPase activity in intact yeast mitochondria as well as Triton X-100-solubilized ATPase preparations. The K(i) for apoptolidin was 4-5 microM. The selectivity of apoptolidin in the NCI-60 cell line panel was found to correlate well with that of several known anti-fungal natural products that inhibit the eukaryotic mitochondrial F0F1-ATP synthase.Although the anti-fungal activities of macrolide inhibitors of the mitochondrial F0F1-ATP synthase such as oligomycin, ossamycin and cytovaricin are well-documented, their unusual selectivity toward certain cell types is not widely appreciated. The recent discovery of apoptolidin, followed by the demonstration that it is an inhibitor of the mitochondrial F0F1-ATP synthase, highlights the potential relevance of these natural products as small molecules to modulate apoptotic pathways. The mechanistic basis for selective cytotoxicity of mitochondrial ATP synthase inhibitors is discussed.

    View details for Web of Science ID 000167275800008

    View details for PubMedID 11182320

  • Understanding and exploiting the mechanistic basis for selectivity of polyketide inhibitors of F0F1-ATPase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Salomon, A. R., Voehringer, D. W., Herzenberg, L. A., Khosla, C. 2000; 97 (26): 14766-14771

    Abstract

    Recently, a family of polyketide inhibitors of F(0)F(1)-ATPase, including apoptolidin, ossamycin, and oligomycin, were shown to be among the top 0.1% most cell line selective cytotoxic agents of 37, 000 molecules tested against the 60 human cancer cell lines of the National Cancer Institute. Many cancer cells maintain a high level of anaerobic carbon metabolism even in the presence of oxygen, a phenomenon that is historically known as the Warburg effect. A mechanism-based strategy to sensitize such cells to this class of potent small molecule cytotoxic agents is presented. These natural products inhibit oxidative phosphorylation by targeting the mitochondrial F(0)F(1) ATP synthase. Evaluation of gene expression profiles in a panel of leukemias revealed a strong correlation between the expression level of the gene encoding subunit 6 of the mitochondrial F(0)F(1) ATP synthase (known to be the binding site of members of this class of macrolides) and their sensitivity to these natural products. Within the same set of leukemia cell lines, comparably strong drug-gene correlations were also observed for the genes encoding two key enzymes involved in central carbon metabolism, pyruvate kinase, and aspartate aminotransferase. We propose a simple model in which the mitochondrial apoptotic pathway is activated in response to a shift in balance between aerobic and anaerobic ATP biosynthesis. Inhibitors of both lactate formation and carbon flux through the Embden-Meyerhof pathway significantly sensitized apoptolidin-resistant tumors to this drug. Nine different cell lines derived from human leukemias and melanomas, and colon, renal, central nervous system, and ovarian tumors are also sensitized to killing by apoptolidin.

    View details for Web of Science ID 000165993700138

    View details for PubMedID 11121076

  • Natural product biosynthesis: A new interface between enzymology and medicine JOURNAL OF ORGANIC CHEMISTRY Khosla, C. 2000; 65 (24): 8127-8133

    View details for Web of Science ID 000165548900001

    View details for PubMedID 11101363

  • Cloning, nucleotide sequence, and heterologous expression of the biosynthetic gene cluster for R1128, a non-steroidal estrogen receptor antagonist - Insights into an unusual priming mechanism JOURNAL OF BIOLOGICAL CHEMISTRY Marti, T., Hu, Z. H., Pohl, N. L., Shah, A. N., Khosla, C. 2000; 275 (43): 33443-33448

    Abstract

    R1128 substances are anthraquinone natural products that were previously reported as non-steroidal estrogen receptor antagonists with in vitro and in vivo potency approaching that of tamoxifen. From a biosynthetic viewpoint, these polyketides possess structurally interesting features such as an unusual primer unit that are absent in the well studied anthracyclic and tetracyclic natural products. The entire R1128 gene cluster was cloned and expressed in Streptomyces lividans, a genetically well developed heterologous host. In addition to R1128C, a novel optically active natural product, designated HU235, was isolated. Nucleotide sequence analysis of the biosynthetic gene cluster revealed genes encoding two ketosynthases, a chain length factor, an acyl transferase, three acetyl-CoA carboxylase subunits, two cyclases, two oxygenases, an amidase, and remarkably, two acyl carrier proteins. Feeding studies indicate that the unusual 4-methylvaleryl side chain of R1128C is derived from valine. Together with the absence of a dedicated ketoreductase, dehydratase, or enoylreductase within the R1128 gene cluster, this suggests a functional link between fatty acid biosynthesis and R1128 biosynthesis in the engineered host. Specifically, we propose that the R1128 synthase recruits four subunits from the endogenous fatty acid synthase during the biosynthesis of this family of pharmacologically significant natural products.

    View details for Web of Science ID 000090104600038

    View details for PubMedID 10931852

  • Substrate specificity of the loading didomain of the erythromycin polyketide synthase BIOCHEMISTRY Lau, J., Cane, D. E., Khosla, C. 2000; 39 (34): 10514-10520

    Abstract

    The priming of many modular polyketide synthases is catalyzed by a loading acyltransferase-acyl carrier protein (AT(L)-ACP(L)) didomain which initiates polyketide biosynthesis by transferring a primer unit to the ketosynthase domain of the first module. Because the AT(L) domain influences the choice of the starter unit incorporated into the polyketide backbone, its specificity is of considerable interest. The AT(L)-ACP(L) didomain of the 6-deoxyerythronolide B synthase (DEBS) was functionally expressed in Escherichia coli. Coexpression of the Sfp phosphopantetheinyl transferase from Bacillus subtilis in E. coli leads to efficient posttranslational modification of the ACP(L) domain with a phosphopantetheine moiety. Competition experiments were performed with the holo-protein to determine the relative rates of incorporation of a variety of unnatural substrates in the presence of comparable concentrations of labeled acetyl-CoA. Our results showed that the loading didomain of DEBS can accept a surprisingly broad range of substrates, although it exhibits a preference for unbranched alkyl chain substrates over branched alkyl chain, polar, aromatic, and charged substrates. In particular, its tolerance toward acetyl- and butyryl-CoA is unexpectedly strong. The studies described here present an attractive prototype for the expression, analysis, and engineering of acyltransferase domains in modular polyketide synthases.

    View details for DOI 10.1021/bi000602v

    View details for Web of Science ID 000089066600019

    View details for PubMedID 10956042

  • Directed transfer of large DNA fragments between Streptomyces species APPLIED AND ENVIRONMENTAL MICROBIOLOGY Hu, Z. H., Hopwood, D. A., Khosla, C. 2000; 66 (5): 2274-2277

    Abstract

    The biosynthesis of complex natural products in bacteria is invariably encoded within large gene clusters. Although this facilitates the cloning of such gene clusters, their heterologous expression in genetically amenable hosts remains a challenging problem, principally due to the difficulties associated with manipulating large DNA fragments. Here we describe a new method for the directed transfer of a gene cluster from one Streptomyces species to another. The method takes advantage of tra gene-mediated conjugal transfer of chromosomal DNA between actinomycetes. As proof of principle, we demonstrate transfer of the entire approximately 22-kb actinorhodin gene cluster, and also the high-frequency cotransfer of two loci that are 150 to 200 kb apart, from Streptomyces coelicolor to an engineered derivative of Streptomyces lividans.

    View details for Web of Science ID 000086805500080

    View details for PubMedID 10788417

  • Mechanistic analysis of a type II polyketide synthase. Role of conserved residues in the beta-ketoacyl synthase-chain length factor heterodimer BIOCHEMISTRY Dreier, J., Khosla, C. 2000; 39 (8): 2088-2095

    Abstract

    Type II polyketide synthases (PKSs) are a family of multienzyme systems that catalyze the biosynthesis of polyfunctional aromatic natural products such as actinorhodin, frenolicin, tetracenomycin, and doxorubicin. A central component in each of these systems is the beta-ketoacyl synthase-chain length factor (KS-CLF) heterodimer. In the presence of an acyl carrier protein (ACP) and a malonyl-CoA:ACP malonyl transferase (MAT), this enzyme synthesizes a polyketide chain of defined length from malonyl-CoA. We have investigated the role of the actinorhodin KS-CLF in priming, elongation, and termination of its octaketide product by subjecting the wild-type enzyme and selected mutants to assays that probe key steps in the overall catalytic cycle. Under conditions reflecting steady-state turnover of the PKS, a unique acyl-ACP intermediate is detected that carries a long, possibly full-length, acyl chain. This species cannot be synthesized by the C169S, H309A, K341A, and H346A mutants of the KS, all of which are blocked in early steps in the PKS catalytic cycle. These four residues are universally conserved in all known KSs. Malonyl-ACP alone is sufficient for kinetically and stoichiometrically efficient synthesis of polyketides by the wild-type KS-CLF, but not by heterodimers that carry the mutations listed above. Among these mutants, C169S is an efficient decarboxylase of malonyl-ACP, but the H309A, K341A, and H346A mutants are unable to catalyze decarboxylation. Transfer of label from [(14)C]malonyl-ACP to the nucleophile at position 169 in the KS can be detected for the wild-type enzyme and for the C169S and K341A mutants, but not for the H309A mutant and only very weakly for the H346A mutant. A model is proposed for decarboxylative priming and extension of a polyketide chain by the KS, where C169 and H346 form a catalytic dyad for acyl chain attachment, H309 positions the malonyl-ACP in the active site and supports carbanion formation by interacting with the thioester carbonyl, and K341 enhances the rate of malonyl-ACP decarboxylation via electrostatic interaction. Our data also suggest that the ACP and the KS dissociate after each C-C bond forming event, and that the newly extended acyl chain is transferred back from the ACP pantetheine to the KS cysteine before dissociation can occur. Chain termination is most likely the rate-limiting step in polyketide biosynthesis. Within the act CLF, neither the universally conserved S145 residue nor Q171, which aligns with the active site cysteine of the ketosynthase, is essential for PKS activity. The results described here provide a basis for a better understanding of the catalytic cycle of type II PKSs and fatty acid synthases.

    View details for Web of Science ID 000085640500022

    View details for PubMedID 10684659

  • Role of linkers in communication between protein modules CURRENT OPINION IN CHEMICAL BIOLOGY Gokhale, R. S., Khosla, C. 2000; 4 (1): 22-27

    Abstract

    Multidomain proteins are common in a variety of cellular processes. Their domains are interconnected through short stretches of amino acid residues referred to as linkers. Recent studies on many systems have provided compelling evidence that linkers are more than simple covalent connectors. They also perform the important task of establishing communication between the different functional modules that exist within such proteins.

    View details for Web of Science ID 000085289500003

    View details for PubMedID 10679375

  • Heterologous expression, purification, reconstitution and kinetic analysis of an extended type II polyketide synthase CHEMISTRY & BIOLOGY Zawada, R. J., Khosla, C. 1999; 6 (9): 607-615

    Abstract

    Polyketide synthases (PKSs) are bacterial multienzyme systems that synthesize a broad range of natural products. The 'minimal' PKS consists of a ketosynthase, a chain length factor, an acyl carrier protein and a malonyl transferase. Auxiliary components (ketoreductases, aromatases and cyclases are involved in controlling the oxidation level and cyclization of the nascent polyketide chain. We describe the heterologous expression and reconstitution of several auxiliary PKS components including the actinorhodin ketoreductase (act KR), the griseusin aromatase/cyclase (gris ARO/CYC), and the tetracenomycin aromatase/cyclase (tcm ARO/CYC).The polyketide products of reconstituted act and tcm PKSs were identical to those identified in previous in vivo studies. Although stable protein-protein interactions were not detected between minimal and auxiliary PKS components, kinetic analysis revealed that the extended PKS comprised of the act minimal PKS, the act KR and the gris ARO/CYC had a higher turnover number than the act minimal PKS plus the act KR or the act minimal PKS alone. Adding the tcm ARO/CYC to the tcm minimal PKS also increased the overall rate.Until recently the principal strategy for functional analysis of PKS subunits was through heterologous expression of recombinant PKSs in Streptomyces. Our results corroborate the implicit assumption that the product isolated from whole-cell systems is the dominant product of the PKS. They also suggest that an intermediate is channeled between the various subunits, and pave the way for more detailed structural and mechanistic analysis of these multienzyme systems.

    View details for Web of Science ID 000082542000005

    View details for PubMedID 10467128

  • A host-vector system for analysis and manipulation of rifamycin polyketide biosynthesis in Amycolatopsis mediterranei MICROBIOLOGY-SGM Hu, Z. H., Hunziker, D., Hutchinson, C. R., Khosla, C. 1999; 145: 2335-2341

    Abstract

    Modular polyketide synthases (PKSs) are a large family of multifunctional enzymes responsible for the biosynthesis of numerous bacterial natural products such as erythromycin and rifamycin. Advanced genetic analysis of these remarkable systems is often seriously hampered by the large size (>40 kb) of PKS gene clusters, and, notwithstanding their considerable fundamental and biotechnological significance, by the lack of suitable methods for engineering non-selectable modifications in chromosomally encoded PKS genes. The development of a facile host-vector strategy for genetic engineering of the rifamycin PKS in the producing organism, Amycolatopsis mediterranei S699, is described here. The genes encoding all 10 modules of the rifamycin PKS were replaced with a hygromycin-resistance marker gene. In a similar construction, only the first six modules of the PKS were replaced. The deletion hosts retained the ability to synthesize the primer unit 3-amino-5-hydroxybenzoic acid (AHBA), as judged by co-synthesis experiments with a mutant strain lacking AHBA synthase activity. Suicide plasmids carrying a short fragment from the 5' flanking end of the engineered deletion, an apramycin-resistance marker gene, and suitably engineered PKS genes could be introduced via electroporation into the deletion hosts, resulting in the integration of PKS genes and biosynthesis of a reporter polyketide in quantities comparable to those produced by the wild-type organism. Since this strategy for engineering recombinant PKSs in A. mediterranei requires only a selectable single crossover and eliminates the need for tedious non-selectable double-crossover experiments, it makes rifamycin PKS an attractive target for extensive genetic manipulation.

    View details for Web of Science ID 000082829000016

    View details for PubMedID 10517586

  • Kinetic analysis of the actinorhodin aromatic polyketide synthase JOURNAL OF BIOLOGICAL CHEMISTRY Dreier, J., Shah, A. N., Khosla, C. 1999; 274 (35): 25108-25112

    Abstract

    Type II polyketide synthases (PKSs) are bacterial multienzyme systems that catalyze the biosynthesis of a broad range of natural products. A core set of subunits, consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and possibly a malonyl CoA:ACP transacylase (MAT) forms a "minimal" PKS. They generate a poly-beta-ketone backbone of a specified length from malonyl-CoA derived building blocks. Here we (a) report on the kinetic properties of the actinorhodin minimal PKS, and (b) present further data in support of the requirement of the MAT. Kinetic analysis showed that the apoACP is a competitive inhibitor of minimal PKS activity, demonstrating the importance of protein-protein interactions between the polypeptide moiety of the ACP and the remainder of the minimal PKS. In further support of the requirement of MAT for PKS activity, two new findings are presented. First, we observe hyperbolic dependence of PKS activity on MAT concentration, saturating at very low amounts (half-maximal rate at 19.7 +/- 5.1 nM). Since MAT can support PKS activity at less than 1/100 the typical concentration of the ACP and ketosynthase/chain length factor components, it is difficult to rule out the presence of trace quantities of MAT in a PKS reaction mixture. Second, an S97A mutant was constructed at the nucleophilic active site of the MAT. Not only can this mutant protein support PKS activity, it is also covalently labeled by [(14)C]malonyl-CoA, demonstrating that the serine nucleophile (which has been the target of PMSF inhibition in earlier studies) is dispensible for MAT activity in a Type II PKS system.

    View details for Web of Science ID 000082193400090

    View details for PubMedID 10455191

  • Dissecting and exploiting intermodular communication in polyketide synthases SCIENCE Gokhale, R. S., Tsuji, S. Y., Cane, D. E., Khosla, C. 1999; 284 (5413): 482-485

    Abstract

    Modular polyketide synthases catalyze the biosynthesis of medicinally important natural products through an assembly-line mechanism. Although these megasynthases display very precise overall selectivity, we show that their constituent modules are remarkably tolerant toward diverse incoming acyl chains. By appropriate engineering of linkers, which exist within and between polypeptides, it is possible to exploit this tolerance to facilitate the transfer of biosynthetic intermediates between unnaturally linked modules. This protein engineering strategy also provides insights into the evolution of modular polyketide synthases.

    View details for Web of Science ID 000079792200046

    View details for PubMedID 10205055

  • Tolerance and specificity of recombinant 6-methylsalicyclic acid synthase. Metabolic engineering Richardson, M. T., Pohl, N. L., Kealey, J. T., Khosla, C. 1999; 1 (2): 180-187

    Abstract

    6-Methylsalicylic acid synthase (MSAS), a fungal polyketide synthase from Penicillium patulum, is perhaps the simplest polyketide synthase that embodies several hallmarks of this family of multifunctional enzymes--a large multidomain protein, a high degree of specificity toward acetyl-CoA and malonyl-CoA substrates, chain length control, and regiospecific ketoreduction. MSAS has recently been functionally expressed in Escherichia coli and Saccharomyces cerevisiae, leading to the engineered biosynthesis of 6-methylsalicylic acid in these hosts. These developments have set the stage for detailed mechanistic studies of this model system.A three--step purification procedure was developed to obtain >95% pure MSAS from extracts of E. coli. As reported earlier for the enzyme isolated from P. patulum, the recombinant enzyme produced 6-methylsalicylic acid (a reduced tetraketide) in the presence of acetyl-CoA, malonyl-CoA, and NADPH, but triacetic acid lactone (an unreduced triketide) in the absence of NADPH. Consistent with this observation, point mutations in the highly conserved nucleotide-binding motif of the ketoreductase domain also led to production of triacetic acid lactone in vivo. The enzyme showed some tolerance toward nonnatural primer units including propionyl- and butyryl-CoA, but was incapable of incorporating extender units from (R, S)-methylmalonyl-CoA. Interestingly, MSAS readily accepted the N-acetylcysteamine (NAC) analog of malonyl-CoA as a substrate.NAC thioesters are simple, cost-effective analogs of CoA thioester substrates, and therefore provide a facile strategy for probing the molecular recognition features of polyketide synthases using unnatural building blocks. The ability to produce 4-hydroxy-6-methyl-2-pyrone in both E. coli and yeast illustrates the feasibility of metabolic engineering of these hosts to produce unnatural polyketides. Finally, the abundant source of recombinant MSAS described here provides an opportunity to study this fascinating model system using a combination of structural, mechanistic, and mutagenesis approaches.

    View details for PubMedID 10935930

  • Dissecting the role of acyltransferase domains of modular polyketide synthases in the choice and stereochemical fate of extender units BIOCHEMISTRY Lau, J., Fu, H., Cane, D. E., Khosla, C. 1999; 38 (5): 1643-1651

    Abstract

    Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), are large multifunctional enzyme complexes that are organized into modules, where each module carries the domains needed to catalyze the condensation of an extender unit onto a growing polyketide chain. Each module also dictates the stereochemistry of the chiral centers introduced into the backbone during the chain elongation process. Here we used domain mutagenesis to investigate the role of the acyl transferase (AT) domains of individual modules in the choice and stereochemical fate of extender units. Our results indicate that the AT domains of DEBS do not influence epimerization of the (2S)-methylmalonyl-CoA extender units. Hence, stereochemical control of the methyl-branched centers generated by DEBS most likely resides in the ketosynthase (KS) domains of the individual modules. In contrast, several recent studies have demonstrated that extender unit specificity can be altered by AT domain substitution. In some of these examples, the resulting polyketide was produced at considerably lower titers than the corresponding natural product. We analyzed one such attenuated mutant of DEBS, in which the methylmalonyl transferase domain of module 2 was replaced with a malonyl transferase domain. As reported earlier, the resulting PKS produced only small quantities of the expected desmethyl analogue of 6-deoxyerythronolide B. However, when the same hybrid module was placed as the terminal module in a truncated 2-module PKS, it produced nearly normal quantities of the expected desmethyl triketide lactone. These results illustrate the limits to modularity of these multifunctional enzymes. To dissect the role of specific amino acids in controlling AT substrate specificity, we exchanged several segments of amino acids between selected malonyl and methylmalonyl transferases, and found that a short (23-35 amino acid) C-terminal segment present in all AT domains is the principal determinant of their substrate specificity. Interestingly, its length and amino acid sequence vary considerably among the known AT domains. We therefore suggest that the choice of extender units by the PKS modules is influenced by a "hypervariable region", which could be manipulated via combinatorial mutagenesis to generate novel AT domains possessing relaxed or altered substrate specificity.

    View details for Web of Science ID 000078836700030

    View details for PubMedID 9931032

  • Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase CHEMISTRY & BIOLOGY Gokhale, R. S., Hunziker, D., Cane, D. E., Khosla, C. 1999; 6 (2): 117-125

    Abstract

    Polyketides are important compounds with antibiotic and anticancer activities. Several modular polyketide synthases (PKSs) contain a terminal thioesterase (TE) domain probably responsible for the release and concomitant cyclization of the fully processed polyketide chain. Because the TE domain influences qualitative aspects of product formation by engineered PKSs, its mechanism and specificity are of considerable interest.The TE domain of the 6-deoxyerythronolide B synthase was overexpressed in Escherichia coli. When tested against a set of N-acetyl cysteamine thioesters the TE domain did not act as a cyclase, but showed significant hydrolytic specificity towards substrates that mimic important features of its natural substrate. Also the overall rate of polyketide chain release was strongly enhanced by a covalent connection between the TE domain and the terminal PKS module (by as much as 100-fold compared with separate TE and PKS 'domains').The inability of the TE domain alone to catalyze cyclization suggests that macrocycle formation results from the combined action of the TE domain and a PKS module. The chain-length and stereochemical preferences of the TE domain might be relevant in the design and engineered biosynthesis of certain novel polyketides. Our results also suggest that the TE domain might loop back to catalyze the release of polyketide chains from both terminal and pre-terminal modules, which may explain the ability of certain naturally occurring PKSs, such as the picromycin synthase, to generate both 12-membered and 14-membered macrolide antibiotics.

    View details for Web of Science ID 000082564800008

    View details for PubMedID 10021418

  • Tolerance and specificity of polyketide synthases ANNUAL REVIEW OF BIOCHEMISTRY Khosla, C., Gokhale, R. S., Jacobsen, J. R., Cane, D. E. 1999; 68: 219-253

    Abstract

    Polyketide synthases catalyze the assembly of complex natural products from simple precursors such as propionyl-CoA and methylmalonyl-CoA in a biosynthetic process that closely parallels fatty acid biosynthesis. Like fatty acids, polyketides are assembled by successive decarboxylative condensations of simple precursors. But whereas the intermediates in fatty acid biosynthesis are fully reduced to generate unfunctionalized alkyl chains, the intermediates in polyketide biosynthesis may be only partially processed, giving rise to complex patterns of functional groups. Additional complexity arises from the use of different starter and chain extension substrates, the generation of chiral centers, and further functional group modifications, such as cyclizations. The structural and functional modularity of these multienzyme systems has raised the possibility that polyketide biosynthetic pathways might be rationally reprogrammed by combinatorial manipulation. An essential prerequisite for harnessing this biosynthetic potential is a better understanding of the molecular recognition features of polyketide synthases. Within this decade, a variety of genetic, biochemical, and chemical investigations have yielded insights into the tolerance and specificity of several architecturally different polyketide synthases. The results of these studies, together with their implications for biosynthetic engineering, are summarized in this review.

    View details for Web of Science ID 000082693200008

    View details for PubMedID 10872449

  • Precursor-directed biosynthesis of 12-ethyl erythromycin BIOORGANIC & MEDICINAL CHEMISTRY Jacobsen, J. R., Keatinge-Clay, A. T., Cane, D. E., Khosla, C. 1998; 6 (8): 1171-1177

    Abstract

    A precursor-directed method for the biosynthesis of novel 6-deoxyerythronolide B derivatives has been extended to allow alteration of the functionality at C-12. We recently described a simple and practical method for harnessing the biosynthetic potential of the erythromycin pathway to generate novel molecules of natural product-like complexity by feeding designed synthetic molecules to an engineered mutant strain having an altered 6-deoxyerythronolide B synthase (DEBS). Our initial applications of this technique focused on alteration of the ethyl side chain of 6-dEB (C14-C15). We now report the extension of this approach to modification of the C-12 substituent. An appropriately designed substrate is shown to incorporate into 6-dEB biosynthesis, yielding a 6-dEB analogue bearing a 12-ethyl group. This 6-dEB analogue is a substrate for post-polyketide tailoring enzymes, and is converted into the corresponding analogue of erythromycin C. These results show that many of the downstream active sites are tolerant of this unnatural functionality and suggest that a wide variety of erythromycin derivatives should be accessible by this approach or by total biosynthesis via genetic engineering.

    View details for Web of Science ID 000075803700005

    View details for PubMedID 9784859

  • Spontaneous priming of a downstream module in 6-deoxyerythronolide B synthase leads to polyketide biosynthesis BIOCHEMISTRY Jacobsen, J. R., Cane, D. E., Khosla, C. 1998; 37 (14): 4928-4934

    Abstract

    Modular polyketide synthases such as 6-deoxyerythronolide B synthase (DEBS) catalyze the biosynthesis of structurally complex natural products by repetitive condensation of simple carboxylic acid monomers. The synthase can be divided into groups of domains, called "modules", each of which is responsible for one cycle of chain extension and processing. The modular nature of these enzymes suggests that the biosynthetic pathway might be rationally reprogrammed by manipulation of synthases at the domain level. Although, several examples of successful engineering of DEBS have been reported, a critical issue which has not been well-studied is the tolerance of "downstream" active sites to nonnatural substrates. Here, we report that the terminal modules of DEBS, which normally process highly functionalized intermediates, are competent to carry out their natural functions on smaller, more simple substrates. Expressed in the absence of other DEBS proteins, the DEBS3 protein, which normally carries out the final two extension cycles in the synthesis of 6-deoxyerythronolide B (6-dEB), is spontaneously primed with a C3 carboxylic acid. This substrate is then extended through two condensation cycles to form a triketide. Tolerance of the "shortened" intermediates in the biosynthesis of this triketide, in combination with results reported elsewhere [Jacobsen, J. R., Hutchinson, C. R., Cane, D. E., and Khosla, C. (1997) Science 277, 367-369], suggests that relaxed substrate specificity may be a common feature of modular polyketide synthases. Interestingly, priming of DEBS3 appears to proceed, not by acyltransfer from propionyl-CoA, but by decarboxylation of an enzyme-bound methylmalonyl extender unit. This is the second example of decarboxylative priming within DEBS [see also Pieper, R., Gokhale, R. S., Luo, G., Cane, D. E., and Khosla, C. (1997) Biochemistry 36, 1846-1851] and suggests that, in the absence of an acceptable primer (or transferred intermediate), decarboxylative priming of ketosynthase domains may be a general property of modular polyketide synthases.

    View details for Web of Science ID 000073279100029

    View details for PubMedID 9538011

  • Functional orientation of the acyltransferase domain in a module of the erythromycin polyketide synthase BIOCHEMISTRY Gokhale, R. S., Lau, J., Cane, D. E., Khosla, C. 1998; 37 (8): 2524-2528

    Abstract

    Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex and medicinally important natural products. These large multienzymes are organized into a series of functional units known as modules. Each dimeric module contains two catalytically independent clusters of active sites homologous to those of vertebrate fatty acid synthases. Earlier studies have shown that modules consist of head-to-tail homodimers in which ketosynthase (KS) and acyl carrier protein (ACP) domains are contributed by opposite subunits to form a catalytic center. Here, we probe the functional topology of the acyltransferase (AT) domain which transfers the methylmalonyl moiety of methylmalonyl-CoA onto the phosphopantetheine arm of the ACP domain. Using a bimodular derivative of DEBS, the AT domain of module 2 (AT2) was inactivated by site-directed mutagenesis. Heterodimeric protein pairs were generated in vitro between the inactivated AT2 (AT2 degrees) polypeptide and an inactive KS1 (KS1 degrees) or KS2 (KS2 degrees) protein. Both of these hybrid proteins supported polyketide synthesis, suggesting that AT2 can perform its function from either subunit. The apparent catalytic rate constants for each of the two hybrid protein pairs, KS1 degrees/AT2 degrees and KS2 degrees/AT2 degrees, were identical, indicating that no significant kinetic preference exists for a particular AT2-ACP2 combination. These results suggest that the AT domain can be shared between the two clusters of active sites within the same dimeric module. Such a novel structural organization might provide a functional advantage for the efficient biosynthesis of polyketides.

    View details for Web of Science ID 000072299900049

    View details for PubMedID 9485401

  • Purification and in vitro reconstitution of the essential protein components of an aromatic polyketide synthase BIOCHEMISTRY Carreras, C. W., Khosla, C. 1998; 37 (8): 2084-2088

    Abstract

    A minimal set of proteins which catalyze the synthesis of aromatic polketides from malonyl CoA has been purified and partially characterized. Plasmid-encoded actinorhodin (act) ketosynthase/chain-length factor (KS/CLF) complex was purified from Streptomyces coelicolor CH999/pSEK38, and assayed with purified aromatic PKS holo-ACPs which were overproduced and purified from Escherichia coli and phosphopantetheinylated in vitro using purified E. coli holo-ACP synthase. When highly purified preparations of KS/CLF, and holo-ACP failed to catalyze polyketide biosynthesis, a fourth protein was sought and purified from the S. coelicolor CH999 host on the basis of its ability to complement KS, CLF, and holo-ACP in polyketide synthesis. N-terminal sequencing identified this protein as the fatty acid synthase (fabD) malonyl CoA:ACP transacylase (MAT), recruited from primary metabolism. A alpha2/beta2 structure was shown for the act KS/CLF complex, and three malonyl-enzyme biosynthetic intermediates were identified, defining an escorted path followed by malonyl groups en route from CoA to polyketide.

    View details for Web of Science ID 000072299900002

    View details for PubMedID 9518007

  • New directions in metabolic engineering CURRENT OPINION IN CHEMICAL BIOLOGY Jacobsen, J. R., Khosla, C. 1998; 2 (1): 133-137

    Abstract

    Metabolic engineering is a rapidly evolving field. The term typically refers to the genetic modification of cellular biochemistry to introduce new properties or modify existing ones. Recent progress in genetics, molecular biology, microbiology and chemistry are driving advances in this field. Many well-studied areas continue to yield exciting results and new problems and technologies are constantly being developed.

    View details for Web of Science ID 000072701000017

    View details for PubMedID 9667907

  • Utilization of enzymatically phosphopantetheinylated acyl carrier proteins and acetyl-acyl carrier proteins by the actinorhodin polyketide synthase BIOCHEMISTRY Carreras, C. W., Gehring, A. M., Walsh, C. T., Khosla, C. 1997; 36 (39): 11757-11761

    Abstract

    The functional reconstitution of two purified proteins of an aromatic polyketide synthase pathway, the acyl carrier protein (ACP) and holo-ACP synthase (ACPS), is described. Holo-ACPs were enzymatically synthesized from coenzyme A and apo-ACPs using Escherichia coli ACPS. Frenolicin and granaticin holo-ACPs formed in this manner were shown to be fully functional together with the other components of the minimal actinorhodin polyketide synthase (act PKS), resulting in synthesis of the same aromatic polyketides as those formed by the act PKS in vivo. ACPS also catalyzed the transfer of acetyl-, propionyl-, butyryl-, benzoyl-, phenylacetyl-, and malonylphosphopantetheines to apo-ACPs from their corresponding coenzyme As, as detected by electrophoresis and/or mass spectrometry. A steady state kinetic study showed that acetyl-coenzyme A is as efficient an ACPS substrate as coenzyme A, with kcat and Km values of 20 min-1 and 25 microM, respectively. In contrast to acetyl-coenzyme A, enzymatically synthesized acetyl-ACPs were shown to be efficient substrates for the act PKS, indicating that acetyl-ACP is a chemically competent intermediate of aromatic polyketide biosynthesis. Together, these methods provide a valuable tool for dissecting the mechanisms and molecular recognition features of polyketide biosynthesis.

    View details for Web of Science ID A1997XY95300023

    View details for PubMedID 9305965

  • Precursor-directed biosynthesis of erythromycin analogs by an engineered polyketide synthase SCIENCE Jacobsen, J. R., Hutchinson, C. R., Cane, D. E., Khosla, C. 1997; 277 (5324): 367-369

    Abstract

    A genetic block was introduced in the first condensation step of the polyketide biosynthetic pathway that leads to the formation of 6-deoxyerythronolide B (6-dEB), the macrocyclic precursor of erythromycin. Exogenous addition of designed synthetic molecules to small-scale cultures of this null mutant resulted in highly selective multimilligram production of unnatural polyketides, including aromatic and ring-expanded variants of 6-dEB. Unexpected incorporation patterns were observed, illustrating the catalytic versatility of modular polyketide synthases. Further processing of some of these scaffolds by postpolyketide enzymes of the erythromycin pathway resulted in the generation of novel antibacterials with in vitro potency comparable to that of their natural counterparts.

    View details for Web of Science ID A1997XL35800045

    View details for PubMedID 9219693

  • Domain analysis of the molecular recognition features of aromatic polyketide synthase subunits JOURNAL OF BIOLOGICAL CHEMISTRY Zawada, R. J., Khosla, C. 1997; 272 (26): 16184-16188

    Abstract

    Bacterial aromatic polyketide synthases (PKSs) are a family of homologous multienzyme assemblies that catalyze the biosynthesis of numerous polyfunctional aromatic natural products. In the absence of direct insights into their structures, the use of gene fusions can be a powerful tool for understanding the structural basis for their properties. A series of truncated and hybrid proteins were constructed and analyzed within a family of PKS subunits, designated aromatases/cyclases (ARO/CYCs). When expressed alone, neither the N-terminal nor the C-terminal domain of the actinorhodin (act) or the griseusin (gris) ARO/CYC exhibited substantial aromatase activity. However, in the presence of each other, the half proteins were active. Furthermore, analysis of a set of hybrid proteins derived from the act and gris ARO/CYCs allowed us to localize the chain length dependence of this aromatase activity to their N-terminal domains. Unexpectedly, however, when the C-terminal domain of the gris ARO/CYC was expressed in a context where aromatase activity was absent, it could modulate the chain length specificity of the tetracenomycin (tcm) minimal PKS, leading to the formation of a novel 18-carbon product in addition to the expected 20-carbon one. It was also found that monodomain ARO/CYCs such as tcmN cannot substitute for the the N-terminal domain of didomain ARO/CYCs, even though they exhibit high sequence similarity with the N-terminal domain. Together, these results illustrate the utility of protein engineering approaches for dissecting the structure-function relationships of PKS subunits and for the generation of mutant alleles with novel biosynthetic properties.

    View details for Web of Science ID A1997XG01900021

    View details for PubMedID 9195917

  • Purification and characterization of bimodular and trimodular derivatives of the erythromycin polyketide synthase BIOCHEMISTRY Pieper, R., Gokhale, R. S., Luo, G. L., Cane, D. E., Khosla, C. 1997; 36 (7): 1846-1851

    Abstract

    Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex and medicinally important natural products. DEBS is a dimeric protein complex that consists of three large multidomain polypeptide chains, DEBS 1, DEBS 2, and DEBS 3. In turn, each polypeptide includes two modules, where one module is responsible for a single round of condensation and associated reduction reactions. A hybrid protein comprised of the first two modules of DEBS fused to a thioesterase domain (DEBS 1 + TE) was purified to homogeneity in a fully active form (Kcat = 4.8 min-1). Synthesis of the anticipated triketide lactone required the presence of (2RS)-methylmalonyl-CoA and NADPH. When available, propionyl-CoA is the preferred source of primer units. However, in its absence the enzyme can derive primer units via decarboxylation of a methylmalonyl extender. The two subunits of an engineered trimodular derivative of DEBS, DEBS 1 and module 3 of DEBS 2 linked to the TE domain (module 3 + TE), were also individually purified and reconstituted to produce the expected tetraketide lactone in vitro (Kcat = 0.23 min-1). The considerably lower specific activity of this trimodular PKS relative to its bimodular counterpart presumably reflects inefficient association between DEBS 1 and module 3 + TE. As expected, module 3 + TE could be efficiently cross-linked as a homodimer. In contrast, no cross-links were detectable between modules 2 and 3, even though biosynthesis of the tetraketide requires transient interactions to occur between these two modules. Since module 3 only contains the minimal set of active sites required in a module (a ketosynthase, an acyltransferase, and an acyl carrier protein domain) and is the first active unimodular protein to be purified to homogeneity, it represents an attractive target for future biophysical and structural studies.

    View details for Web of Science ID A1997WJ05800031

    View details for PubMedID 9048569

  • Harnessing the Biosynthetic Potential of Modular Polyketide Synthases. Chemical reviews Khosla, C. 1997; 97 (7): 2577-2590

    View details for PubMedID 11851472

  • A functional chimeric modular polyketide synthase generated via domain replacement CHEMISTRY & BIOLOGY Bedford, D., Jacobsen, J. R., Luo, G. L., Cane, D. E., Khosla, C. 1996; 3 (10): 827-831

    Abstract

    Modular polyketide synthases (PKSs), such as 6-deoxyerythronolide B synthase (DEBS), are large multifunctional enzymes that catalyze the biosynthesis of structurally complex and medically important natural products. Active sites within these assemblies are organized into 'modules', such that each module catalyzes the stereospecific addition of a new monomer onto a growing polyketide chain and also sets the reduction level of the beta-carbon atom of the resulting intermediate. The core of each module is made up of a 'reductive segment', which includes all, some, or none of a set of ketoreductase (KR), dehydratase, and enoylreductase domains, in addition to a large interdomain region which lacks overt function but may contribute to structural stability and inter-domain dynamics within modules. The highly conserved organization of reductive segments within modules suggests that they might be able to function in unnatural contexts to generate novel organic molecules.To investigate domain substitution as a method for altering PKS function, a chimeric enzyme was engineered. Using a bimodular derivative of DEBS (DEBS1+TE), the reductive segment of module 2, which includes a functional KR, was replaced with its homolog from module 3 of DEBS, which contains a (naturally occurring) nonfunctional KR. A recombinant strain expressing the chimeric gene produced the predicted ketolactone with a yield (35 %) comparable to that of a control strain in which the KR2 domain was retained but mutationally inactivated.These results demonstrate considerable structural tolerance within an important segment found in virtually every PKS module. The domain boundaries defined here could be exploited for the construction of numerous loss-of-function and possibly even gain-of-function mutants within this remarkable family of multifunctional enzymes.

    View details for Web of Science ID A1996VR14500006

    View details for PubMedID 8939701

  • Evidence for two catalytically independent clusters of active sites in a functional modular polyketide synthase BIOCHEMISTRY Kao, C. M., Pieper, R., Cane, D. E., Khosla, C. 1996; 35 (38): 12363-12368

    Abstract

    Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex and medicinally important natural products. These large multifunctional enzymes are organized into "modules", where each module contains active sites homologous to those of higher eucaryotic fatty acid synthases (FASs). Like FASs, modular PKSs are known to be dimers. Here we provide functional evidence for the existence of two catalytically independent clusters of active sites within a modular PKS. In three bimodular derivatives of DEBS, the ketosynthase domain of module 1 (KS-1) or module 2 (KS-2) or the acyl carrier protein domain of module 2 (ACP-2) was inactivated via site-directed mutagenesis. As expected, the purified proteins were unable to catalyze polyketide synthesis (although the KS-1 mutant could convert a diketide thioester into the predicted triketide lactone). Remarkably however, the KS-1/KS-2 and the KS-2/ACP-2 mutant pairs could efficiently complement each other and catalyze polyketide formation. In contrast, the KS-1 and ACP-2 mutants did not complement each other. On the basis of these and other results, a model is proposed in which the individual modules of a PKS dimer form head-to-tail homodimers, thereby generating two equivalent and independent clusters of active sites for polyketide biosynthesis. Specifically, each subunit contributes half of the KS and ACP domains in each cluster. A similar complementation approach should also be useful in dissecting the organization of the remaining types of active sites within this family of multienzyme assemblies. Finally, blocked systems, such as the KS-1 mutant described here, present a new strategy for the noncompetitive conversion of unnatural substrates into polyketides by modular PKSs.

    View details for Web of Science ID A1996VJ27000018

    View details for PubMedID 8823171

  • Generation of polyketide libraries via combinatorial biosynthesis TRENDS IN BIOTECHNOLOGY Khosla, C., Zawada, R. J. 1996; 14 (9): 335-341

    Abstract

    Polyketides are a family of structurally complex natural products that include a number of important pharmaceuticals. Motivated by the value of these natural products, there has been much research focused on developing guidelines for engineering polyketide synthases (PKSs) to generate novel polyketides. Recent studies have provided interesting insights into the enzymatic specificity of the polyketide synthesis pathway, and have demonstrated that various PKSs can be genetically manipulated to synthesize 'unnatural' polyketide natural products. In this article, we discuss the synthesis of polyketides and polyketide libraries by combinatorial biosynthesis.

    View details for Web of Science ID A1996VF73800007

    View details for PubMedID 8818287

  • Engineered biosynthesis of novel polyketides: Regiospecific methylation of an unnatural substrate by the tcmO O-methyltransferase BIOCHEMISTRY Fu, H., Alvarez, M. A., Khosla, C., Bailey, J. E. 1996; 35 (21): 6527-6532

    Abstract

    TcmO is an O-methyltransferase that methylates the C-8 hydroxyl to Tcm B3, a four-ring aromatic intermediate in the tetracenomycin biosynthetic pathway of Streptomyces glaucescens. The gene encoding this enzyme was expressed in Streptomyces coelicolor CH999 together with the actinorhodin polyketide synthase (PKS) gene cluster, which is responsible for the biosynthesis of 3,8-dihydroxy-methylanthraquinone carboxylic acid (DMAC) and its decarboxylated analog, aloesaponarin. The resulting recombinant strain produced approximately equal quantities of aloesaponarin and a new product but no DMAC. Spectroscopic analysis revealed that the novel polyketide was the 3-O-methylated analog of DMAC. An in vitro radioisotopic assay was developed for tcmO. The enzyme requires S-adenosylmethionine as a co-substrate. It has a Km of 3 microM and a kcat of 2.7 min-1 for DMAC. A series of monocyclic, bicyclic, and tricyclic aromatic compounds were also tested as candidate substrates in vitro. Remarkably, none was modified by tcmO within detectable limits of the assay. Together, these results highlight the interesting molecular recognition features of this enzyme. On one hand, there appears to be some flexibility in the number and structures of unreactive rings, since both Tcm and B3 and DMAC are good substrates. However, 6-methylsalicylic acid, a monocyclic analog of the reactive ring, is not recognized by the enzyme. Likewise, neither aloesaponarin (which only differs from DMAC in the reactive ring) nor carminic acid (which only differs in the distal nonreactive ring) is modified. Thus, the binding energy for the tcmO-catalyzed methyl transfer appears to involve significant contributions from both the aromaticity and the functionality of polycyclic substrates.

    View details for Web of Science ID A1996UN28800002

    View details for PubMedID 8639600

  • Combinatorial chemistry and biology: An opportunity for engineers CURRENT OPINION IN BIOTECHNOLOGY Khosla, C. 1996; 7 (2): 219-222

    Abstract

    Within the past two years, the burgeoning field of combinatorial chemistry and biology has witnessed major advances in both technologies and applications. New ideas have emerged, and continue to be sought, with regard to library design, construction, and analysis. The highly multi-disciplinary nature of the field, together with its need for a systems-based view of pertinent challenges and problems, makes it an ideal area for biochemical engineers.

    View details for Web of Science ID A1996UE90500016

    View details for PubMedID 8791337

  • Erythromycin biosynthesis: Kinetic studies on a fully active modular polyketide synthase using natural and unnatural substrates BIOCHEMISTRY Pieper, R., EBERTKHOSLA, S., Cane, D., Khosla, C. 1996; 35 (7): 2054-2060

    Abstract

    6-Deoxyerythronolide B synthase (DEBS) is a modular polyketide synthase (PKS) that catalyzes the biosynthesis of the parent macrolide of erythromycin. On the basis of a recently developed cell-free assay (Pieper et al., 1995a) we report the results of steady-state kinetic studies on a modular PKS. A truncated form of DEBS (DEBS 1+TE), in which DEBS 1 is fused to the thioesterase domain from the C-terminal end of DEBS 3, was used for most of these studies. The overall k(cat) for (2S,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxy-n-heptanoic acid delta-lactone (C9-lactone) synthesis is 3.4 min(-1), indicating that the enzyme is at least as active in vitro as in vivo. The apparent K(m) for (2S)-methylmalonyl-CoA consumption by DEBS 1+TE is 24 microM. The catalytic activity of DEBS 1+TE is strongly dependent on the phosphate concentration in the reaction buffer in the range 0-250 mM, suggesting that hydrophobic interactions may be crucial to the assembly of DEBS monomers into a functional complex. Although DEBS 1+TE can convert acetyl-, propionyl-, or butyryl-CoA into the corresponding C8-, C9-, and C10-lactones (Pieper et al., 1995b), it has a 32-fold preference for a propionate primer over an acetate primer and a 7.5-fold preference for a propionate primer over a butyrate primer. In the absence of any added primer unit, synthesis can be primed via decarboxylation of methylmalonyl-CoA; under these conditions the overall k(cat) for polyketide synthesis remains unchanged. Decarboxylation of methylmalonyl-CoA is negligible in the presence of saturating concentrations of propionyl-CoA but competes with the priming of the enzyme by acetyl-CoA or butyryl-CoA. The k(cat) for 6-deoxyerythronolide B synthesis by the complete DEBS is 0.5 min(-1). Under these assay conditions, the C9-lactone is also produced as an abortive chain elongation product with a k(cat) of 0.23 min(-1), presumably due to inefficient assembly of the multimeric protein complex involving DEBS 1, 2, and 3. Together, these results provide the first comprehensive kinetic insights into a fully active modular PKS.

    View details for Web of Science ID A1996TW34500002

    View details for PubMedID 8652546

  • Antibiotic activity of polyketide products derived from combinatorial biosynthesis: Implications for directed evolution MOLECULAR DIVERSITY Fu, H., Khosla, C. 1996; 1 (2): 121-124

    Abstract

    A library of over 100 polyketides, generated via combinatorial cloning of genes encoding subunits of aromatic polyketide synthases, was screened for molecules capable of inhibiting the growth of gram-positive bacteria. A total of 26 polyketides, with varying levels of antibiotic activity in filter-disk assays, were purified. Most bioactive polyketides were produced as relatively minor compounds (< 1 mg/l), although two major anthraquinones, with yields in the range of 10-100 mg/l, were also identified and structurally characterized. When tested against Bacillus subtilis 168 beta, they were found to cause a 50% reduction in colony-forming units at concentrations of 20 and 300 micrograms/ml, respectively. We speculate that many of the minor (and possibly more potent) bioactive polyketides are synthesized via nonspecific enzymatic modifications of shunt products derived from engineered polyketide synthase pathways. If so, then these 'fortuitous' pathways should be amenable to further rationally guided manipulation. Our results support the notion that combinatorial biosynthesis can be used to generate novel, biologically active molecules. They also point to the feasibility of designing mutagenesis selection experiments aimed at the directed evolution of organic molecules with desirable pharmaceutical properties.

    View details for Web of Science ID A1996VV71400006

    View details for PubMedID 9237201

  • Engineering of novel polyketides - Progress and prospects ENZYME ENGINEERING XIII Kramer, P. J., Khosla, C. 1996; 799: 32-45

    View details for Web of Science ID 000070949000006

    View details for PubMedID 8958070

  • CELL-FREE SYNTHESIS OF POLYKETIDES BY RECOMBINANT ERYTHROMYCIN POLYKETIDE SYNTHASES NATURE Pieper, R., Luo, G. L., Cane, D. E., Khosla, C. 1995; 378 (6554): 263-266

    Abstract

    Modular polyketide synthases (PKSs) are complex multi-enzyme proteins that catalyse the bacterial biosynthesis of many pharmaceutically useful polyketides. The PKSs are organized into a series of modules, each containing the active catalytic sites required for one step in the synthesis process. Here we report a method for cell-free enzymatic synthesis of 6-deoxyerythronolide B (6-dEB), the parent molecule of the antibiotic erythromycin A, using recombinant 6-deoxyerythronolide B synthase (DEBS), a modular PKS with at least 28 distinct active sites. We have also synthesized in vitro a triketide lactone by using a truncated mutant of DEBS. The availability of such cell-free synthetic routes will allow direct investigation of the structural and mechanistic basis for the unusual combination of high substrate specificity and tolerance to genetic reprogramming found in this enzyme family.

    View details for Web of Science ID A1995TE85800043

    View details for PubMedID 7477343

  • EXPRESSION OF A FUNCTIONAL FUNGAL POLYKETIDE SYNTHASE IN THE BACTERIUM STREPTOMYCES-COELICOLOR A3(2) JOURNAL OF BACTERIOLOGY Bedford, D. J., Schweizer, E., Hopwood, D. A., Khosla, C. 1995; 177 (15): 4544-4548

    Abstract

    The multifunctional 6-methylsalicylic acid synthase gene from Penicillium patulum was engineered for regulated expression in Streptomyces coelicolor. Production of significant amounts of 6-methylsalicylic acid by the recombinant strain was proven by nuclear magnetic resonance spectroscopy. These results suggest that it is possible to harness the molecular diversity of eukaryotic polyketide pathways by heterologous expression of biosynthetic genes in an easily manipulated model bacterial host in which prokaryotic aromatic and modular polyketide synthase genes are already expressed and recombined.

    View details for Web of Science ID A1995RL82800050

    View details for PubMedID 7635840

  • RATIONAL DESIGN OF AROMATIC POLYKETIDE NATURAL-PRODUCTS BY RECOMBINANT ASSEMBLY OF ENZYMATIC SUBUNITS NATURE McDaniel, R., EBERTKHOSLA, S., Hopwood, D. A., Khosla, C. 1995; 375 (6532): 549-554

    Abstract

    Recent advances in understanding of bacterial aromatic polyketide biosynthesis allow the development of a set of design rules for the rational manipulation of chain synthesis, reduction of keto groups and early cyclization steps by genetic engineering. The concept of rational design is illustrated by the preparation of Streptomyces strains that produce two new polyketides by expression of combinations of appropriate enzymatic subunits from naturally occurring polyketide synthases. The potential for generating molecular diversity within this class of molecules by genetic engineering is enormous.

    View details for Web of Science ID A1995RD28700041

    View details for PubMedID 7791871

  • COMBINATORIAL BIOSYNTHESIS OF UNNATURAL NATURAL-PRODUCTS - THE POLYKETIDE EXAMPLE CHEMISTRY & BIOLOGY TSOI, C. J., Khosla, C. 1995; 2 (6): 355-362

    Abstract

    Multi-enzyme systems, such as those involved in the biosynthesis of polyketides, typically catalyze several distinct reactions that are combined in different ways to generate diverse natural products. The variability available in such systems has not been fully harnessed from nature. It may therefore be possible to create 'unnatural' natural products, which may be as structurally diverse and medicinally valuable as existing natural products, using combinatorial biosynthesis.

    View details for Web of Science ID A1995RH72200003

    View details for PubMedID 9383437

  • Engineered biosynthesis of novel polyketides: evidence for temporal, but not regiospecific, control of cyclization of an aromatic polyketide precursor. Chemistry & biology Fu, H., Hopwood, D. A., Khosla, C. 1994; 1 (4): 205-210

    Abstract

    Aromatic polyketide synthases (PKSs) catalyze the formation and cyclization of polyketide chains of variable lengths, generating a family of compounds of proven medical significance. Initial control over the regiospecificity of cyclization is believed to be exercised by the minimal PKS, composed of the three essential components for polyketide biosynthesis, which catalyzes an intramolecular aldol condensation towards the middle of the chain. Subsequent cyclization reactions are either catalyzed by additional components of the PKS, or occur in the absence of specific catalysts.Structural and biosynthetic studies on SEK4b, a novel octaketide product of a minimal PKS, revealed an unusual cyclization pattern. The first cyclization (an aldol condensation) occurs at the methyl end of the unreduced polyketide backbone precursor. This is followed by hemiketal formation and lactonization. The overall structure of SEK4b is similar to that of SEK4, a previously-identified product of the same genetically-engineered strain, differing only in the positions of a methyl and a pyrone group around a common fused-ring system. The biosynthetic pathways of the two molecules are quite different, however. The yield of SEK4b relative to SEK4 is much higher in the absence of PKS components (aromatases and cyclases) acting later in the pathway.In this cyclization pathway, the regiospecificity of cyclization is not directly controlled by the minimal PKS. Instead, we propose that the enzyme influences cyclization by controlling the timing of chain release. Chain release and cyclization may be concurrent with synthesis. Other PKS subunits appear to stabilize the complex of the PKS with the nascent chain, preventing premature release.

    View details for PubMedID 9383392

  • ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES - ACT(VII) AND ACT(IV) GENES ENCODE AROMATASE AND CYCLASE ENZYMES, RESPECTIVELY JOURNAL OF THE AMERICAN CHEMICAL SOCIETY McDaniel, R., EBERTKHOSLA, S., Hopwood, D. A., Khosla, C. 1994; 116 (24): 10855-10859
  • ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES - INFLUENCE OF A DOWNSTREAM ENZYME ON THE CATALYTIC SPECIFICITY OF A MINIMAL AROMATIC POLYKETIDE SYNTHASE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA McDaniel, R., EBERTKHOSLA, S., Fu, H., Hopwood, D. A., Khosla, C. 1994; 91 (24): 11542-11546

    Abstract

    To identify the minimum set of polyketide synthase (PKS) components required for in vivo biosynthesis of aromatic polyketides, combinations of genes encoding subunits of three different aromatic PKSs--act from Streptomyces coelicolor A3(2) (an actinorhodin producer), fren from Streptomyces roseofulvus (a frenolicin and nanaomycin producer), and tcm from Streptomyces glaucescens (a tetracenomycin producer)--were expressed in a recently developed Streptomyces host-vector system. The "minimal" components (ketosynthase/putative acyltransferase, chain length-determining factor, and acyl carrier protein) were produced with and without a functional polyketide ketoreductase and/or cyclase, and the polyketide products of these recombinant strains were structurally characterized. Several previously identified polyketides were isolated in addition to two previously unidentified polyketides, dehydromutactin and SEK 15b, described here. The results proved that the act cyclase is not required for the biosynthesis of several aberrantly cyclized products that have been previously reported. They are also consistent with earlier conclusions that the minimal PKS controls chain length as well as the regiospecificity of the first cyclization and that it can do so in the absence of both a ketoreductase and a cyclase. However, the ability of the minimal tcm PKS to synthesize two different singly cyclized intermediates suggests that it is unable to accurately control the course of this reaction by itself. In the presence of a downstream enzyme, the flux through one branch of the cyclization pathway increases relative to the other. We propose that these alternative specificities may be due to the ability of downstream enzymes to associate with the minimal PKS and to selectively inhibit a particular branch of the cyclization pathway.

    View details for Web of Science ID A1994PU28500053

    View details for PubMedID 7972098

  • ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES - STEREOCHEMICAL COURSE OF 2 REACTIONS CATALYZED BY A POLYKETIDE SYNTHASE BIOCHEMISTRY Fu, H., McDaniel, R., Hopwood, D. A., Khosla, C. 1994; 33 (31): 9321-9326

    Abstract

    A genetically engineered strain expressing the essential components of the tetracenomycin polyketide synthase (tcm PKS) along with the actinorhodin ketoreductase (act KR) was found to produce two new (diastereomeric) aromatic polyketides, designated RM20b and RM20c, in addition to RM20, whose structure was reported earlier [McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1993) Science 262, 1546-1550]. Spectroscopic and in vivo isotopic labeling analysis of RM20b and RM20c revealed that their polyketide backbones were identical to that of RM20 with respect to chain length, regiospecificity of ketoreduction, and regiospecificity of the first intramolecular aldol condensation. This is consistent with earlier predictions that the essential components of the PKS--a bifunctional ketosynthase/acyltransferase, a chain length determining factor, and an acyl carrier protein--are responsible for controlling each of these features of the polyketide backbone [McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1993) Science 262, 1546-1550; McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1993) J. Am. Chem. Soc. 115, 11671-11675; Fu, H., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1994) J. Am. Chem. Soc. 116, 4166-4170]. In addition, however, RM20b and RM20c possess two unusual features. In both molecules the hydroxyls on sp3 C-9 and C-7 of the first six-membered ring, which arise as a result of ketoreduction and aldol condensation, respectively, are intact, rather than being lost via dehydration. Furthermore, the relative yield of RM20b (in which these hydroxyls are syn) is 7-fold greater than that of RM20c (in which they are anti).(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1994PB76300036

    View details for PubMedID 8049233

  • ENGINEERED BIOSYNTHESIS OF A COMPLETE MACROLACTONE IN A HETEROLOGOUS HOST SCIENCE Kao, C. M., Katz, L., Khosla, C. 1994; 265 (5171): 509-512

    Abstract

    Macrocyclic polyketides have been subjects of great interest in synthetic and biosynthetic chemistry because of their structural complexity and medicinal activities. With expression of the entire 6-deoxyerythronolide B synthase (DEBS) (10,283 amino acids) in a heterologous host, substantial quantities of 6-deoxyerythronolide B (6dEB), the aglycone of the macrolide antibiotic erythromycin, and 8,8a-deoxyoleandolide, a 14-membered lactone ring identical to 6dEB except for a methyl group side chain in place of an ethyl unit, were synthesized in Streptomyces coelicolor. The biosynthetic strategy utilizes a genetic approach that facilitates rapid structural manipulation of DEBS or other modular polyketide synthases (PKSs), including those found in actinomycetes with poorly developed genetic methods. From a technological viewpoint, this approach should allow the rational design of biosynthetic products and may eventually lead to the generation of diverse polyketide libraries by means of combinatorial cloning of naturally occurring and mutant PKS modules.

    View details for Web of Science ID A1994NY21600025

    View details for PubMedID 8036492

  • ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES - DISSECTION OF THE CATALYTIC SPECIFICITY OF THE ACT KETOREDUCTASE JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Fu, H., EBERTKHOSLA, S., Hopwood, D. A., Khosla, C. 1994; 116 (10): 4166-4170
  • EFFICIENT SAMPLING OF PROTEIN-SEQUENCE SPACE FOR MULTIPLE MUTANTS BIO-TECHNOLOGY Caren, R., Morkeberg, R., Khosla, C. 1994; 12 (5): 517-520

    Abstract

    We describe here a method capable of generating a very large population of multiple mutants, the size of which is primarily limited by volume constraints. This method, referred to as recombination-enhanced mutagenesis, combines the power of in vitro mutagenesis with the high frequencies of in vivo recombination that can be achieved using single-stranded transduction systems. The recombination frequency between two mutations separated by as little as 19 amino acids is 0.02; this frequency approaches a value of 0.1 for mutations separated by more than 38 amino acids. Up to 10(8) independent recombinants were generated in 1 ml of an E. coli culture, and this number scales linearly (or better) with increasing volume. To prove the method's effectiveness, we applied it to the problem of reverting multiple mutants of mouse dihydrofolate reductase, which could not be reverted using mutagenesis alone. Thus, given an appropriate screen or selection scheme, recombination-enhanced mutagenesis is well-suited for addressing a range of combinatorially complex problems, such as antigen recognition, enzyme catalysis, protein folding, and transport/transduction across biomembranes.

    View details for Web of Science ID A1994NJ86300029

    View details for PubMedID 7764711

  • ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES - MANIPULATION AND ANALYSIS OF AN AROMATIC POLYKETIDE SYNTHASE WITH UNPROVED CATALYTIC SPECIFICITIES JOURNAL OF THE AMERICAN CHEMICAL SOCIETY McDaniel, R., EBERTKHOSLA, S., Hopwood, D. A., Khosla, C. 1993; 115 (25): 11671-11675
  • ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES SCIENCE McDaniel, R., EBERTKHOSLA, S., Hopwood, D. A., Khosla, C. 1993; 262 (5139): 1546-1550

    Abstract

    Polyketide synthases (PKSs) are multifunctional enzymes that catalyze the biosynthesis of a huge variety of carbon chains differing in their length and patterns of functionality and cyclization. Many polyketides are valuable therapeutic agents. A Streptomyces host-vector system has been developed for efficient construction and expression of recombinant PKSs. Using this expression system, several novel compounds have been synthesized in vivo in significant quantities. Characterization of these metabolites has provided new insights into key features of actinomycete aromatic PKS specificity. Thus, carbon chain length is dictated, at least in part, by a protein that appears to be distinctive to this family of PKSs, whereas the acyl carrier proteins of different PKSs can be interchanged without affecting product structure. A given ketoreductase can recognize and reduce polyketide chains of different length; this ketoreduction always occurs at the C-9 position. The regiospecificity of the first cyclization of the nascent polyketide chain is either determined by the ketoreductase, or the chain-extending enzymes themselves. However, the regiospecificity of the second cyclization is determined by a distinct cyclase, which can discriminate between substrates of different chain lengths.

    View details for Web of Science ID A1993MK32900033

    View details for PubMedID 8248802

  • TARGETED GENE REPLACEMENTS IN A STREPTOMYCES POLYKETIDE SYNTHASE GENE-CLUSTER - ROLE FOR THE ACYL CARRIER PROTEIN MOLECULAR MICROBIOLOGY Khosla, C., EBERTKHOSLA, S., Hopwood, D. A. 1992; 6 (21): 3237-3249

    Abstract

    A methodology was developed to construct any desired chromosomal mutation in the gene cluster that encodes the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2). A positive selection marker (resistance gene) is first introduced by double crossing-over into the chromosomal site of interest by use of an unstable delivery plasmid. This marker is subsequently replaced by the desired mutant allele via a second high-frequency double recombination event. The technology has been used to: (i) explore the significance of translational coupling between two adjacent PKS genes; (ii) prove that the acyl carrier protein (ACP) encoded by a gene in the cluster is necessary for the function of the actinorhodin PKS; (iii) provide genetic evidence supporting the hypothesis that serine 42 is the site of phosphopantetheinylation in the ACP of the actinorhodin PKS; and (iv) demonstrate that this ACP can be replaced by a Saccharopolyspora fatty acid synthase ACP to generate an active hybrid PKS.

    View details for Web of Science ID A1992JW88800014

    View details for PubMedID 1453961

Conference Proceedings


  • Engineered biosynthesis of polyketides in heterologous hosts Rude, M. A., Khosla, C. PERGAMON-ELSEVIER SCIENCE LTD. 2004: 4693-4701
  • Evolutionally guided enzyme design Khosla, C., Caren, R., Kao, C. M., McDaniel, R., Wang, S. W. JOHN WILEY & SONS INC. 1996: 122-128

    Abstract

    A combination of "rational" and "irrational" strategies for the creation of enzymes with novel properties is proving to be a powerful concept in the field of enzyme engineering. Guided by principles of physical organic chemistry, rational design strategies are used to identify suitable target enzymes and to choose appropriate molecular biological methods for engineering purposes. In contrast, irrational (or random) strategies are centered around the biological paradigm of stochastic molecular evolution. As illustrated in this review, such a hybrid approach is particularly useful for the design of new modular enzymes. (c) 1996 John Wiley & Sons, Inc.

    View details for Web of Science ID A1996VH41300013

    View details for PubMedID 18629858

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