Dylan Dodd

All Publications

• Perspective: Dietaryiomarkers of Intake and Exposure-Exploration with Omics Approaches. Advances in nutrition (Bethesda, Md.) Maruvada, P., Lampe, J. W., Wishart, D. S., Barupal, D., Chester, D. N., Dodd, D., Djoumbou-Feunang, Y., Dorrestein, P. C., Dragsted, L. O., Draper, J., Duffy, L. C., Dwyer, J. T., Emenaker, N. J., Fiehn, O., Gerszten, R. E., B Hu, F., Karp, R. W., Klurfeld, D. M., Laughlin, M. R., Little, A. R., Lynch, C. J., Moore, S. C., Nicastro, H. L., O'Brien, D. M., Ordovas, J. M., Osganian, S. K., Playdon, M., Prentice, R., Raftery, D., Reisdorph, N., Roche, H. M., Ross, S. A., Sang, S., Scalbert, A., Srinivas, P. R., Zeisel, S. H. 2019

  Abstract

  While conventional nutrition research has yielded biomarkers such as doubly labeled water for energy metabolism and 24-h urinary nitrogen for protein intake, a critical need exists for additional, equally robust biomarkers that allow for objective assessment of specific food intake and dietary exposure. Recent advances in high-throughput MS combined with improved metabolomics techniques and bioinformatic tools provide new opportunities for dietary biomarker development. In September 2018, the NIH organized a 2-d workshop to engage nutrition and omics researchers and explore the potential of multiomics approaches in nutritional biomarker research. The current Perspective summarizes key gaps and challenges identified, as well as the recommendations from the workshop that could serve as a guide for scientists interested in dietary biomarkers research. Topics addressed included study designs for biomarker development, analytical and bioinformatic considerations, and integration of dietary biomarkers with other omics techniques. Several clear needs were identified, including larger controlled feeding studies, testing a variety of foods and dietary patterns across diverse populations, improved reporting standards to support study replication, more chemical standards covering a broader range of food constituents and human metabolites, standardized approaches for biomarker validation, comprehensive and accessible food composition databases, a common ontology for dietary biomarker literature, and methodologic work on statistical procedures for intake biomarker discovery. Multidisciplinary research teams with appropriate expertise are critical to moving forward the field of dietary biomarkers and producing robust, reproducible biomarkers that can be used in public health and clinical research.

  View details for DOI 10.1093/advances/nmz075

  View details for PubMedID 31386148

• Clinical utility of an ultrasensitive urinary free cortisol assay by tandem mass spectrometry STEROIDS Luo, A., El Gierari, E. M., Nally, L. M., Sturmer, L. R., Dodd, D., Shi, R. 2019; 146: 65?69

  View details for DOI 10.1016/j.steroids.2019.03.014

  View details for Web of Science ID 000467508700008

• Clinical Utility of an Ultrasensitive Urinary Free Cortisol Assay by Tandem Mass Spectrometry. Steroids Luo, A., El Gierari, E. T., Nally, L. M., Sturmer, L. R., Dodd, D., Shi, R. Z. 2019

  Abstract

  24 h urinary free cortisol measurement is a clinically important first-line screening test for Cushing's syndrome (CS). Tandem mass spectrometry (LC-MS/MS) assays have superior sensitivity and specificity compared to immunoassays. Our goal was to improve and validate a LC-MS/MS method to measure urinary free cortisol in both adult and pediatric patients and to characterize its clinical diagnostic performance of CS by chart review.We improved a LC-MS/MS method previously reported for urinary free cortisol to be able to measure urinary and salivary cortisol in the same batch for increased efficiency. The sample preparation was by liquid-liquid extraction using dichloromethane followed by stepwise washing with acidic, basic and neutral solutions. The assay's analytical performance was characterized, and a retrospective patient chart review was conducted to evaluate the assay's clinical performance in diagnosing CS.The LC-MS/MS assay demonstrated enhanced sensitivity and was linear within an analytical measurement range of 10-10,000 ng/dL. Assay accuracy was satisfactory as determined by spike and recovery studies and highly correlated with a reference LC-MS/MS method. The assay's clinical diagnostic sensitivity and specificity in detecting CS was 96% and 91%, respectively, when compared to a urinary cortisol excretion of at least 50 µg/24 h.The improved LC-MS/MS method is both sensitive and specific with enhanced analytical performance and clinical diagnostic utility to screen for CS. The clinical diagnostic sensitivity and specificity were superior based on retrospective patient chart review.

  View details for PubMedID 30951757

• A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites. Nature Dodd, D., Spitzer, M. H., Van Treuren, W., Merrill, B. D., Hryckowian, A. J., Higginbottom, S. K., Le, A., Cowan, T. M., Nolan, G. P., Fischbach, M. A., Sonnenburg, J. L. 2017; 551 (7682): 648?52

  Abstract

  The human gut microbiota produces dozens of metabolites that accumulate in the bloodstream, where they can have systemic effects on the host. Although these small molecules commonly reach concentrations similar to those achieved by pharmaceutical agents, remarkably little is known about the microbial metabolic pathways that produce them. Here we use a combination of genetics and metabolic profiling to characterize a pathway from the gut symbiont Clostridium sporogenes that generates aromatic amino acid metabolites. Our results reveal that this pathway produces twelve compounds, nine of which are known to accumulate in host serum. All three aromatic amino acids (tryptophan, phenylalanine and tyrosine) serve as substrates for the pathway, and it involves branching and alternative reductases for specific intermediates. By genetically manipulating C. sporogenes, we modulate serum levels of these metabolites in gnotobiotic mice, and show that in turn this affects intestinal permeability and systemic immunity. This work has the potential to provide the basis of a systematic effort to engineer the molecular output of the gut bacterial community.

  View details for PubMedID 29168502

• Enzymatic mechanism for arabinan degradation and transport in the thermophilic bacterium Caldanaerobius polysaccharolyticus. Applied and environmental microbiology Wefers, D., Dong, J., Abdel-Hamid, A. M., Müller Paul, H., Pereira, G. V., Han, Y., Dodd, D., Baskaran, R., Mayer, B., Mackie, R. I., Cann, I. 2017

  Abstract

  The plant cell wall polysaccharide arabinan is an important supply of arabinose, and unraveling arabinan degrading strategies by microbes is important for understanding its use as a source of energy. Here, we explored the arabinan degrading enzymes in the thermophilic bacterium Caldanaerobius polysaccharolyticus and identified a gene cluster encoding two glycoside hydrolase (GH) family 51 ?-L-arabinofuranosidases (CpAbf51A, CpAbf51B), a GH43 endo-arabinanase (CpAbn43A), a GH27 ?-L-arabinopyranosidase (CpAbp27A), and two GH127 ?-L-arabinofuranosidases (CpAbf127A, CpAbf127B). The genes were expressed as recombinant proteins, and the functions of the purified proteins were determined with pNP-linked sugars and naturally occurring pectin structural elements as substrates. The results demonstrated that CpAbn43A is an endo-arabinanase, while CpAbf51A and CpAbf51B are ?-L-arabinofuranosidases that exhibit diverse substrate specificities, cleaving ?-1,2-, ?-1,3-, and ?-1,5-linkages of purified arabinan-oligosaccharides. Furthermore, both CpAbf127A and CpAbf127B cleaved ?-arabinofuranose residues in complex arabinan side chains, thus providing evidence of the function of this family of enzymes on such polysaccharides. The optimal temperatures of the enzymes ranged between 60 °C and 75 °C, and CpAbf43A and CpAbf51A worked synergistically to release arabinose from branched and debranched arabinan. Furthermore, the hydrolytic activity on branched arabinan oligosaccharides and degradation of pectic substrates by the endo-arabinanase and L-arabinofuranosidases suggested a microbe equipped with diverse activities to degrade complex arabinan in the environment. Based on our functional analyses of the genes in the arabinan degradation cluster and the substrate-binding studies on a component of the cognate transporter system, we propose a model for arabinan degradation and transport by C. polysaccharolyticusImportance Genomic DNA sequencing and bioinformatic analysis allowed for the identification of a gene cluster encoding several proteins predicted to function in arabinan degradation and transport in C. polysaccharolyticus The analysis of the recombinant proteins yielded detailed insights into the putative arabinan metabolism of this thermophilic bacterium. The use of various branched arabinan oligosaccharides provided detailed understanding of the substrate specificities of the enzymes, and allowed assignment of two new GH127 polypeptides as ?-L-arabinofuranosidases, able to degrade pectic substrates, and thus expanding our knowledge on this rare group of glycoside hydrolases. In addition, the enzymes showed synergistic effects for the degradation of arabinans at elevated temperatures. The enzymes characterized from the gene cluster are, therefore, of utility for arabinose production in both the biofuel and food industries.

  View details for DOI 10.1128/AEM.00794-17

  View details for PubMedID 28710263

• Clinical Utility of an Ultrasensitive Late Night Salivary Cortisol Assay by Tandem Mass Spectrometry. Steroids Sturmer, L. R., Dodd, D., Chao, C. S., Shi, R. Z. 2017

  Abstract

  Late night salivary cortisol measurement is a clinically important and convenient screening test for Cushing's syndrome. Tandem mass spectrometry (LC-MS/MS) assays have superior sensitivity and specificity compared to immunoassays. Our goal was to improve a LC-MS/MS method to measure salivary cortisol in both adult and pediatric patients and to characterize its analytical performance by method validation and clinical performance by chart review.We improved a LC-MS/MS method originally developed for urine cortisol to measure low level salivary cortisol. The sample preparation was by liquid-liquid extraction using dichloromethane followed by stepwise washing with acidic, basic and neutral solutions. The assay's analytical performance was characterized and retrospective patient chart review was conducted to evaluate the assay's clinical diagnostic performance.The LC-MS/MS assay showed enhanced functional sensitivity of 10 ng/dL for salivary cortisol and was linear within an analytical measurement range of 10-10,000 ng/dL. Assay accuracy was within 84-120% as determined by recovery studies and correlation with a reference method. Data from healthy adult volunteers was compiled to establish the reference interval for late night salivary cortisol. Patient chart review determined subjects with diagnosis of Cushing's syndrome or disease, and assay's clinical diagnostic sensitivity of 100% and specificity of 92% when the cutoff value was 70 ng/dL.The improved LC-MS/MS method is sensitive and specific with enhanced analytical performance and clinical diagnostic utility for screening Cushing's syndrome. The assay may have broad clinical application due to its high sensitivity and wide dynamic range.

  View details for PubMedID 29197558

• Modulation of a Circulating Uremic Solute via Rational Genetic Manipulation of the Gut Microbiota CELL HOST & MICROBE Devlin, A. S., Marcobal, A., Dodd, D., Nayfach, S., Plummer, N., Meyer, T., Pollard, K. S., Sonnenburg, J. L., Fischbach, M. A. 2016; 20 (6): 709-715

  Abstract

  Renal disease is growing in prevalence and has striking co-morbidities with metabolic and cardiovascular disease. Indoxyl sulfate (IS) is a toxin that accumulates in plasma when kidney function declines and contributes to the progression of chronic kidney disease. IS derives exclusively from the gut microbiota. Bacterial tryptophanases convert tryptophan to indole, which is absorbed and modified by the host to produce IS. Here, we identify a widely distributed family of tryptophanases in the gut commensal Bacteroides and find that deleting this gene eliminates the production of indole in vitro. By altering the status or abundance of the Bacteroides tryptophanase, we can modulate IS levels in gnotobiotic mice and in the background of a conventional murine gut community. Our results demonstrate that it is possible to control host IS levels by targeting the microbiota and suggest a possible strategy for treating renal disease.

  View details for DOI 10.1016/j.chom.2016.10.021

  View details for Web of Science ID 000392843500008

  View details for PubMedID 27916477

  View details for PubMedCentralID PMC5159218

• Your gut microbiome, deconstructed. Nature biotechnology Dodd, D., Tropini, C., Sonnenburg, J. L. 2015; 33 (12): 1238?40

  View details for PubMedID 26650010

• Structural and Biochemical Basis for Mannan Utilization by Caldanaerobius polysaccharolyticus Strain ATCC BAA-17 JOURNAL OF BIOLOGICAL CHEMISTRY Chekan, J. R., Kwon, I. H., Agarwal, V., Dodd, D., Revindran, V., Mackie, R. I., Cann, I., Nair, S. K. 2014; 289 (50): 34965-34977

  Abstract

  Hemicelluloses, the polysaccharide component of plant cell walls, represent one of the most abundant biopolymers in nature. The most common hemicellulosic constituents of softwoods, such as conifers and cycads, are mannans consisting of a 1,4-linked ?-mannopyranosyl main chain with branch decorations. Efforts toward the utilization of hemicellulose for bioconversion into cellulosic biofuels have resulted in the identification of several families of glycoside hydrolases that can degrade mannan. However, effective biofermentation of manno-oligosaccharides is limited by a lack of appropriate uptake route in ethanologenic organisms. Here, we used transcriptome sequencing to gain insights into mannan degradation by the thermophilic anaerobic bacterium Caldanaerobius polysaccharolyticus. The most highly up-regulated genes during mannan fermentation occur in a cluster containing several genes encoding enzymes for efficient mannan hydrolysis as well as a solute-binding protein (CpMnBP1) that exhibits specificity for short mannose polymers but exhibited the flexibility to accommodate branched polysaccharide decorations. Co-crystal structures of CpMnBP1 in complex with mannobiose (1.4-Å resolution) and mannotriose (2.2-Å resolution) revealed the molecular rationale for chain length and oligosaccharide specificity. Calorimetric analysis of several active site variants confirmed the roles of residues critical to the function of CpMnBP1. This work represents the first biochemical characterization of a mannose-specific solute-binding protein and provides a framework for engineering mannan utilization capabilities for microbial fermentation.

  View details for DOI 10.1074/jbc.M114.579904

  View details for Web of Science ID 000346260800045

  View details for PubMedID 25342756

• Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhang, M., Chekan, J. R., Dodd, D., Hong, P., Radlinski, L., Revindran, V., Nair, S. K., Mackie, R. I., Cann, I. 2014; 111 (35): E3708-E3717

  Abstract

  Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT_04215 and BACOVA_04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xylose-configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 Å), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 Å), and the CBM from its homolog in the Prevotella bryantii B14 Xyn10C (1.68 Å) reveal an unanticipated mode for ligand binding. A minimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health.

  View details for DOI 10.1073/pnas.1406156111

  View details for Web of Science ID 000341230800018

  View details for PubMedID 25136124

• Two New Xylanases with Different Substrate Specificities from the Human Gut Bacterium Bacteroides intestinalis DSM 17393 APPLIED AND ENVIRONMENTAL MICROBIOLOGY Hong, P., Iakiviak, M., Dodd, D., Zhang, M., Mackie, R. I., Cann, I. 2014; 80 (7): 2084-2093

  Abstract

  Xylan is an abundant plant cell wall polysaccharide and is a dominant component of dietary fiber. Bacteria in the distal human gastrointestinal tract produce xylanase enzymes to initiate the degradation of this complex heteropolymer. These xylanases typically derive from glycoside hydrolase (GH) families 10 and 11; however, analysis of the genome sequence of the xylan-degrading human gut bacterium Bacteroides intestinalis DSM 17393 revealed the presence of two putative GH8 xylanases. In the current study, we demonstrate that the two genes encode enzymes that differ in activity. The xyn8A gene encodes an endoxylanase (Xyn8A), and rex8A encodes a reducing-end xylose-releasing exo-oligoxylanase (Rex8A). Xyn8A hydrolyzed both xylopentaose (X5) and xylohexaose (X6) to a mixture of xylobiose (X2) and xylotriose (X3), while Rex8A hydrolyzed X3 through X6 to a mixture of xylose (X1) and X2. Moreover, rex8A is located downstream of a GH3 gene (xyl3A) that was demonstrated to exhibit ?-xylosidase activity and would be able to further hydrolyze X2 to X1. Mutational analyses of putative active site residues of both Xyn8A and Rex8A confirm their importance in catalysis by these enzymes. Recent genome sequences of gut bacteria reveal an increase in GH8 Rex enzymes, especially among the Bacteroidetes, indicating that these genes contribute to xylan utilization in the human gut.

  View details for DOI 10.1128/AEM.03176-13

  View details for Web of Science ID 000332840700005

  View details for PubMedID 24463968

• Mutational and Structural Analyses of Caldanaerobius polysaccharolyticus Man5B Reveal Novel Active Site Residues for Family 5 Glycoside Hydrolases PLOS ONE Oyama, T., Schmitz, G. E., Dodd, D., Han, Y., Burnett, A., Nagasawa, N., Mackie, R. I., Nakamura, H., Morikawa, K., Cann, I. 2013; 8 (11)

  Abstract

  CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both ?-1,4-mannosidic and ?-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

  View details for DOI 10.1371/journal.pone.0080448

  View details for Web of Science ID 000327313100089

  View details for PubMedID 24278284

• Reconstitution of a Thermostable Xylan-Degrading Enzyme Mixture from the Bacterium Caldicellulosiruptor bescii APPLIED AND ENVIRONMENTAL MICROBIOLOGY Su, X., Han, Y., Dodd, D., Moon, Y. H., Yoshida, S., Mackie, R. I., Cann, I. K. 2013; 79 (5): 1481-1490

  Abstract

  Xylose, the major constituent of xylans, as well as the side chain sugars, such as arabinose, can be metabolized by engineered yeasts into ethanol. Therefore, xylan-degrading enzymes that efficiently hydrolyze xylans will add value to cellulases used in hydrolysis of plant cell wall polysaccharides for conversion to biofuels. Heterogeneous xylan is a complex substrate, and it requires multiple enzymes to release its constituent sugars. However, the components of xylan-degrading enzymes are often individually characterized, leading to a dearth of research that analyzes synergistic actions of the components of xylan-degrading enzymes. In the present report, six genes predicted to encode components of the xylan-degrading enzymes of the thermophilic bacterium Caldicellulosiruptor bescii were expressed in Escherichia coli, and the recombinant proteins were investigated as individual enzymes and also as a xylan-degrading enzyme cocktail. Most of the component enzymes of the xylan-degrading enzyme mixture had similar optimal pH (5.5 to ?6.5) and temperature (75 to ?90°C), and this facilitated their investigation as an enzyme cocktail for deconstruction of xylans. The core enzymes (two endoxylanases and a ?-xylosidase) exhibited high turnover numbers during catalysis, with the two endoxylanases yielding estimated k(cat) values of ?8,000 and ?4,500 s(-1), respectively, on soluble wheat arabinoxylan. Addition of side chain-cleaving enzymes to the core enzymes increased depolymerization of a more complex model substrate, oat spelt xylan. The C. bescii xylan-degrading enzyme mixture effectively hydrolyzes xylan at 65 to 80°C and can serve as a basal mixture for deconstruction of xylans in bioenergy feedstock at high temperatures.

  View details for DOI 10.1128/AEM.03265-12

  View details for Web of Science ID 000314893300008

  View details for PubMedID 23263957

• Biochemical and Structural Insights into Xylan Utilization by the Thermophilic Bacterium Caldanaerobius polysaccharolyticus JOURNAL OF BIOLOGICAL CHEMISTRY Han, Y., Agarwal, V., Dodd, D., Kim, J., Bae, B., Mackie, R. I., Nair, S. K., Cann, I. K. 2012; 287 (42): 34946-34960

  Abstract

  Hemicellulose is the next most abundant plant cell wall component after cellulose. The abundance of hemicellulose such as xylan suggests that their hydrolysis and conversion to biofuels can improve the economics of bioenergy production. In an effort to understand xylan hydrolysis at high temperatures, we sequenced the genome of the thermophilic bacterium Caldanaerobius polysaccharolyticus. Analysis of the partial genome sequence revealed a gene cluster that contained both hydrolytic enzymes and also enzymes key to the pentose-phosphate pathway. The hydrolytic enzymes in the gene cluster were demonstrated to convert products from a large endoxylanase (Xyn10A) predicted to anchor to the surface of the bacterium. We further use structural and calorimetric studies to demonstrate that the end products of Xyn10A hydrolysis of xylan are recognized and bound by XBP1, a putative solute-binding protein, likely for transport into the cell. The XBP1 protein showed preference for xylo-oligosaccharides as follows: xylotriose > xylobiose > xylotetraose. To elucidate the structural basis for the oligosaccharide preference, we solved the co-crystal structure of XBP1 complexed with xylotriose to a 1.8-? resolution. Analysis of the biochemical data in the context of the co-crystal structure reveals the molecular underpinnings of oligosaccharide length specificity.

  View details for DOI 10.1074/jbc.M112.391532

  View details for Web of Science ID 000309968000008

  View details for PubMedID 22918832

• Biochemical Characterization and Relative Expression Levels of Multiple Carbohydrate Esterases of the Xylanolytic Rumen Bacterium Prevotella ruminicola 23 Grown on an Ester-Enriched Substrate APPLIED AND ENVIRONMENTAL MICROBIOLOGY Kabel, M. A., Yeoman, C. J., Han, Y., Dodd, D., Abbas, C. A., de Bont, J. A., Morrison, M., Cann, I. K., Mackie, R. I. 2011; 77 (16): 5671-5681

  Abstract

  We measured expression and used biochemical characterization of multiple carbohydrate esterases by the xylanolytic rumen bacterium Prevotella ruminicola 23 grown on an ester-enriched substrate to gain insight into the carbohydrate esterase activities of this hemicellulolytic rumen bacterium. The P. ruminicola 23 genome contains 16 genes predicted to encode carbohydrate esterase activity, and based on microarray data, four of these were upregulated >2-fold at the transcriptional level during growth on an ester-enriched oligosaccharide (XOS(FA,Ac)) from corn relative to a nonesterified fraction of corn oligosaccharides (AXOS). Four of the 16 esterases (Xyn10D-Fae1A, Axe1-6A, AxeA1, and Axe7A), including the two most highly induced esterases (Xyn10D-Fae1A and Axe1-6A), were heterologously expressed in Escherichia coli, purified, and biochemically characterized. All four enzymes showed the highest activity at physiologically relevant pH (6 to 7) and temperature (30 to 40°C) ranges. The P. ruminicola 23 Xyn10D-Fae1A (a carbohydrate esterase [CE] family 1 enzyme) released ferulic acid from methylferulate, wheat bran, corn fiber, and XOS(FA,Ac), a corn fiber-derived substrate enriched in O-acetyl and ferulic acid esters, but exhibited negligible activity on sugar acetates. As expected, the P. ruminicola Axe1-6A enzyme, which was predicted to possess two distinct esterase family domains (CE1 and CE6), released ferulic acid from the same substrates as Xyn10D-Fae1 and was also able to cleave O-acetyl ester bonds from various acetylated oligosaccharides (AcXOS). The P. ruminicola 23 AxeA1, which is not assigned to a CE family, and Axe7A (CE7) were found to be acetyl esterases that had activity toward a broad range of mostly nonpolymeric acetylated substrates along with AcXOS. All enzymes were inhibited by the proximal location of other side groups like 4-O-methylglucuronic acid, ferulic acid, or acetyl groups. The unique diversity of carbohydrate esterases in P. ruminicola 23 likely gives it the ability to hydrolyze substituents on the xylan backbone and enhances its capacity to efficiently degrade hemicellulose.

  View details for DOI 10.1128/AEM.05321-11

  View details for Web of Science ID 000293504400014

  View details for PubMedID 21742923

• Xylan degradation, a metabolic property shared by rumen and human colonic Bacteroidetes MOLECULAR MICROBIOLOGY Dodd, D., Mackie, R. I., Cann, I. K. 2011; 79 (2): 292-304

  Abstract

  Microbial inhabitants of the bovine rumen fulfil the majority of the normal caloric requirements of the animal by fermenting lignocellulosic plant polysaccharides and releasing short chain fatty acids that are then metabolized by the host. This process also occurs within the human colon, although the fermentation products contribute less to the overall energy requirements of the host. Mounting evidence, however, indicates that the community structure of the distal gut microbiota is a critical factor that influences the inflammatory potential of the immune system thereby impacting the progression of inflammatory bowel diseases. Non-digestible dietary fibre derived from plant material is highly enriched in the lignocellulosic polysaccharides, cellulose and xylan. Members of the Bacteroidetes constitute a dominant phylum in both the human colonic microbiome and the rumen microbial ecosystem. In the current article, we review recent insights into the molecular mechanisms for xylan degradation by rumen and human commensal members of the Bacteroidetes phylum, and place this information in the context of the physiological and metabolic processes that occur within these complex microbial environments.

  View details for DOI 10.1111/j.1365-2958.2010.07473.x

  View details for Web of Science ID 000286114200004

  View details for PubMedID 21219452

• Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules JOURNAL OF BIOLOGICAL CHEMISTRY Su, X., Agarwal, V., Dodd, D., Bae, B., Mackie, R. I., Nair, S. K., Cann, I. K. 2010; 285 (45): 34665-34676

  Abstract

  Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues that flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.

  View details for DOI 10.1074/jbc.M110.168302

  View details for Web of Science ID 000283659100042

  View details for PubMedID 20739280

• Transcriptomic Analyses of Xylan Degradation by Prevotella bryantii and Insights into Energy Acquisition by Xylanolytic Bacteroidetes JOURNAL OF BIOLOGICAL CHEMISTRY Dodd, D., Moon, Y., Swaminathan, K., Mackie, R. I., Cann, I. K. 2010; 285 (39): 30261-30273

  Abstract

  Enzymatic depolymerization of lignocellulose by microbes in the bovine rumen and the human colon is critical to gut health and function within the host. Prevotella bryantii B(1)4 is a rumen bacterium that efficiently degrades soluble xylan. To identify the genes harnessed by this bacterium to degrade xylan, the transcriptomes of P. bryantii cultured on either wheat arabinoxylan or a mixture of its monosaccharide components were compared by DNA microarray and RNA sequencing approaches. The most highly induced genes formed a cluster that contained putative outer membrane proteins analogous to the starch utilization system identified in the prominent human gut symbiont Bacteroides thetaiotaomicron. The arrangement of genes in the cluster was highly conserved in other xylanolytic Bacteroidetes, suggesting that the mechanism employed by xylan utilizers in this phylum is conserved. A number of genes encoding proteins with unassigned function were also induced on wheat arabinoxylan. Among these proteins, a hypothetical protein with low similarity to glycoside hydrolases was shown to possess endoxylanase activity and subsequently assigned to glycoside hydrolase family 5. The enzyme was designated PbXyn5A. Two of the most similar proteins to PbXyn5A were hypothetical proteins from human colonic Bacteroides spp., and when expressed each protein exhibited endoxylanase activity. By using site-directed mutagenesis, we identified two amino acid residues that likely serve as the catalytic acid/base and nucleophile as in other GH5 proteins. This study therefore provides insights into capture of energy by xylanolytic Bacteroidetes and the application of their enzymes as a resource in the biofuel industry.

  View details for DOI 10.1074/jbc.M110.141788

  View details for Web of Science ID 000281984300062

  View details for PubMedID 20622018

• Comparative Analyses of Two Thermophilic Enzymes Exhibiting both beta-1,4 Mannosidic and beta-1,4 Glucosidic Cleavage Activities from Caldanaerobius polysaccharolyticus JOURNAL OF BACTERIOLOGY Han, Y., Dodd, D., Hespen, C. W., Ohene-Adjei, S., Schroeder, C. M., Mackie, R. I., Cann, I. K. 2010; 192 (16): 4111-4121

  Abstract

  The hydrolysis of polysaccharides containing mannan requires endo-1,4-beta-mannanase and 1,4-beta-mannosidase activities. In the current report, the biochemical properties of two endo-beta-1,4-mannanases (Man5A and Man5B) from Caldanaerobius polysaccharolyticus were studied. Man5A is composed of an N-terminal signal peptide (SP), a catalytic domain, two carbohydrate-binding modules (CBMs), and three surface layer homology (SLH) repeats, whereas Man5B lacks the SP, CBMs, and SLH repeats. To gain insights into how the two glycoside hydrolase family 5 (GH5) enzymes may aid the bacterium in energy acquisition and also the potential application of the two enzymes in the biofuel industry, two derivatives of Man5A (Man5A-TM1 [TM1 stands for truncational mutant 1], which lacks the SP and SLH repeats, and Man5A-TM2, which lacks the SP, CBMs, and SLH repeats) and the wild-type Man5B were biochemically analyzed. The Man5A derivatives displayed endo-1,4-beta-mannanase and endo-1,4-beta-glucanase activities and hydrolyzed oligosaccharides with a degree of polymerization (DP) of 4 or higher. Man5B exhibited endo-1,4-beta-mannanase activity and little endo-1,4-beta-glucanase activity; however, this enzyme also exhibited 1,4-beta-mannosidase and cellodextrinase activities. Man5A-TM1, compared to either Man5A-TM2 or Man5B, had higher catalytic activity with soluble and insoluble polysaccharides, indicating that the CBMs enhance catalysis of Man5A. Furthermore, Man5A-TM1 acted synergistically with Man5B in the hydrolysis of beta-mannan and carboxymethyl cellulose. The versatility of the two enzymes, therefore, makes them a resource for depolymerization of mannan-containing polysaccharides in the biofuel industry. Furthermore, on the basis of the biochemical and genomic data, a molecular mechanism for utilization of mannan-containing nutrients by C. polysaccharolyticus is proposed.

  View details for DOI 10.1128/JB.00257-10

  View details for Web of Science ID 000280406300005

  View details for PubMedID 20562312

• Functional Diversity of Four Glycoside Hydrolase Family 3 Enzymes from the Rumen Bacterium Prevotella bryantii B(1)4 JOURNAL OF BACTERIOLOGY Dodd, D., Kiyonari, S., Mackie, R. I., Cann, I. K. 2010; 192 (9): 2335-2345

  Abstract

  Prevotella bryantii B(1)4 is a member of the phylum Bacteroidetes and contributes to the degradation of hemicellulose in the rumen. The genome of P. bryantii harbors four genes predicted to encode glycoside hydrolase (GH) family 3 (GH3) enzymes. To evaluate whether these genes encode enzymes with redundant biological functions, each gene was cloned and expressed in Escherichia coli. Biochemical analysis of the recombinant proteins revealed that the enzymes exhibit different substrate specificities. One gene encoded a cellodextrinase (CdxA), and three genes encoded beta-xylosidase enzymes (Xyl3A, Xyl3B, and Xyl3C) with different specificities for either para-nitrophenyl (pNP)-linked substrates or substituted xylooligosaccharides. To identify the amino acid residues that contribute to catalysis and substrate specificity within this family of enzymes, the roles of conserved residues (R177, K214, H215, M251, and D286) in Xyl3B were probed by site-directed mutagenesis. Each mutation led to a severely decreased catalytic efficiency without a change in the overall structure of the mutant enzymes. Through amino acid sequence alignments, an amino acid residue (E115) that, when mutated to aspartic acid, resulted in a 14-fold decrease in the k(cat)/K(m) for pNP-beta-d-xylopyranoside (pNPX) with a concurrent 1.1-fold increase in the k(cat)/K(m) for pNP-beta-d-glucopyranoside (pNPG) was identified. Amino acid residue E115 may therefore contribute to the discrimination between beta-xylosides and beta-glucosides. Our results demonstrate that each of the four GH3 enzymes has evolved to perform a specific role in lignopolysaccharide hydrolysis and provide insight into the role of active-site residues in catalysis and substrate specificity for GH3 enzymes.

  View details for DOI 10.1128/JB.01654-09

  View details for Web of Science ID 000276685800006

  View details for PubMedID 20190048

• Thermostable Enzymes as Biocatalysts in the Biofuel Industry ADVANCES IN APPLIED MICROBIOLOGY, VOL 70 Yeoman, C. J., Han, Y., Dodd, D., Schroeder, C. M., Mackie, R. I., Cann, I. K. 2010; 70: 1-55

  Abstract

  Lignocellulose is the most abundant carbohydrate source in nature and represents an ideal renewable energy source. Thermostable enzymes that hydrolyze lignocellulose to its component sugars have significant advantages for improving the conversion rate of biomass over their mesophilic counterparts. We review here the recent literature on the development and use of thermostable enzymes for the depolymerization of lignocellulosic feedstocks for biofuel production. Furthermore, we discuss the protein structure, mechanisms of thermostability, and specific strategies that can be used to improve the thermal stability of lignocellulosic biocatalysts.

  View details for DOI 10.1016/S0065-2164(10)70001-0

  View details for Web of Science ID 000274947600001

  View details for PubMedID 20359453

• Biochemical Analysis of a beta-D-Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in Prevotella ruminicola 23 JOURNAL OF BACTERIOLOGY Dodd, D., Kocherginskaya, S. A., Spies, M. A., Beery, K. E., Abbas, C. A., Mackie, R. I., Cann, I. K. 2009; 191 (10): 3328-3338

  Abstract

  Prevotella ruminicola 23 is an obligate anaerobic bacterium in the phylum Bacteroidetes that contributes to hemicellulose utilization within the bovine rumen. To gain insight into the cellular machinery that this organism elaborates to degrade the hemicellulosic polymer xylan, we identified and cloned a gene predicted to encode a bifunctional xylanase-ferulic acid esterase (xyn10D-fae1A) and expressed the recombinant protein in Escherichia coli. Biochemical analysis of purified Xyn10D-Fae1A revealed that this protein possesses both endo-beta-1,4-xylanase and ferulic acid esterase activities. A putative glycoside hydrolase (GH) family 3 beta-D-glucosidase gene, with a novel PA14-like insertion sequence, was identified two genes downstream of xyn10D-fae1A. Biochemical analyses of the purified recombinant protein revealed that the putative beta-D-glucosidase has activity for pNP-beta-D-xylopyranoside, pNP-alpha-L-arabinofuranoside, and xylo-oligosaccharides; thus, the gene was designated xyl3A. When incubated in combination with Xyn10D-Fae1A, Xyl3A improved the release of xylose monomers from a hemicellulosic xylan substrate, suggesting that these two enzymes function synergistically to depolymerize xylan. Directed mutagenesis studies of Xyn10D-Fae1A mapped the catalytic sites for the two enzymatic functionalities to distinct regions within the polypeptide sequence. When a mutation was introduced into the putative catalytic site for the xylanase domain (E280S), the ferulic acid esterase activity increased threefold, which suggests that the two catalytic domains for Xyn10D-Fae1A are functionally coupled. Directed mutagenesis of conserved residues for Xyl3A resulted in attenuation of activity, which supports the assignment of Xyl3A as a GH family 3 beta-D-xylosidase.

  View details for DOI 10.1128/JB.01628-08

  View details for Web of Science ID 000265625300016

  View details for PubMedID 19304844

• Determinants of Catalytic Power and Ligand Binding in Glutamate Racemase JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Spies, M. A., Reese, J. G., Dodd, D., Pankow, K. L., Blanke, S. R., Baudry, J. 2009; 131 (14): 5274-5284

  Abstract

  Glutamate racemases (EC 5.1.1.3) catalyze the cofactor-independent stereoinversion of D- and L-glutamate and are important for viability in several gram-negative and -positive bacteria. As the only enzyme involved in the stereoinversion of L- to D-glutamate for peptidoglycan biosynthesis, glutamate racemase is an attractive target for the design of antibacterial agents. However, the development of competitive tight-binding inhibitors has been problematic and highly species specific. Despite a number of recent crystal structures of cofactor-independent epimerases and racemases, cocrystallized with substrates or substrate analogues, the source of these enzymes' catalytic power and their ability to acidify the C alpha of amino acids remains unknown. The present integrated computational and experimental study focuses on the glutamate racemase from Bacillus subtilis (RacE). A particular focus is placed on the interaction of the glutamate carbanion intermediate with RacE. Results suggest that the reactive form of the RacE-glutamate carbanion complex, vis-à-vis proton abstraction from C alpha, is significantly different than the RacE-D-glutamate complex on the basis of the crystal structure and possesses dramatically stronger enzyme-ligand interaction energy. In silico and experimental site-directed mutagenesis indicates that the strength of the RacE-glutamate carbanion interaction energy is highly distributed among numerous electrostatic interactions in the active site, rather than being dominated by strong hydrogen bonds. Results from this study are important for laying the groundwork for discovery and design of high-affinity ligands to this class of cofactor-independent racemases.

  View details for DOI 10.1021/ja809660g

  View details for Web of Science ID 000265039000054

  View details for PubMedID 19309142

• Enzymatic deconstruction of xylan for biofuel production. Global change biology. Bioenergy Dodd, D., Cann, I. K. 2009; 1 (1): 2?17

  Abstract

  The combustion of fossil-derived fuels has a significant impact on atmospheric carbon dioxide (CO(2)) levels and correspondingly is an important contributor to anthropogenic global climate change. Plants have evolved photosynthetic mechanisms in which solar energy is used to fix CO(2) into carbohydrates. Thus, combustion of biofuels, derived from plant biomass, can be considered a potentially carbon neutral process. One of the major limitations for efficient conversion of plant biomass to biofuels is the recalcitrant nature of the plant cell wall, which is composed mostly of lignocellulosic materials (lignin, cellulose, and hemicellulose). The heteropolymer xylan represents the most abundant hemicellulosic polysaccharide and is composed primarily of xylose, arabinose, and glucuronic acid. Microbes have evolved a plethora of enzymatic strategies for hydrolyzing xylan into its constituent sugars for subsequent fermentation to biofuels. Therefore, microorganisms are considered an important source of biocatalysts in the emerging biofuel industry. To produce an optimized enzymatic cocktail for xylan deconstruction, it will be valuable to gain insight at the molecular level of the chemical linkages and the mechanisms by which these enzymes recognize their substrates and catalyze their reactions. Recent advances in genomics, proteomics, and structural biology have revolutionized our understanding of the microbial xylanolytic enzymes. This review focuses on current understanding of the molecular basis for substrate specificity and catalysis by enzymes involved in xylan deconstruction.

  View details for DOI 10.1111/j.1757-1707.2009.01004.x

  View details for PubMedID 20431716

  View details for PubMedCentralID PMC2860967

• Functional comparison of the two Bacillus anthracis glutamate racemases JOURNAL OF BACTERIOLOGY Dodd, D., Reese, J. G., Louer, C. R., Ballard, J. D., Spies, M. A., Blanke, S. R. 2007; 189 (14): 5265-5275

  Abstract

  Glutamate racemase activity in Bacillus anthracis is of significant interest with respect to chemotherapeutic drug design, because L-glutamate stereoisomerization to D-glutamate is predicted to be closely associated with peptidoglycan and capsule biosynthesis, which are important for growth and virulence, respectively. In contrast to most bacteria, which harbor a single glutamate racemase gene, the genomic sequence of B. anthracis predicts two genes encoding glutamate racemases, racE1 and racE2. To evaluate whether racE1 and racE2 encode functional glutamate racemases, we cloned and expressed racE1 and racE2 in Escherichia coli. Size exclusion chromatography of the two purified recombinant proteins suggested differences in their quaternary structures, as RacE1 eluted primarily as a monomer, while RacE2 demonstrated characteristics of a higher-order species. Analysis of purified recombinant RacE1 and RacE2 revealed that the two proteins catalyze the reversible stereoisomerization of L-glutamate and D-glutamate with similar, but not identical, steady-state kinetic properties. Analysis of the pH dependence of L-glutamate stereoisomerization suggested that RacE1 and RacE2 both possess two titratable active site residues important for catalysis. Moreover, directed mutagenesis of predicted active site residues resulted in complete attenuation of the enzymatic activities of both RacE1 and RacE2. Homology modeling of RacE1 and RacE2 revealed potential differences within the active site pocket that might affect the design of inhibitory pharmacophores. These results suggest that racE1 and racE2 encode functional glutamate racemases with similar, but not identical, active site features.

  View details for DOI 10.1128/JB.00352-07

  View details for Web of Science ID 000248019700031

  View details for PubMedID 17496086