Autoproteolytic Activation of a Symbiosis-regulated Truffle Phospholipase A(2)
JOURNAL OF BIOLOGICAL CHEMISTRY
2013; 288 (3): 1533-1547
Efficient Electron Transfer in a Protein Network Lacking Specific Interactions
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2011; 133 (42): 16861-16867
Fungal phospholipases are members of the fungal/bacterial group XIV secreted phospholipases A(2) (sPLA(2)s). TbSP1, the sPLA(2) primarily addressed in this study, is up-regulated by nutrient deprivation and is preferentially expressed in the symbiotic stage of the ectomycorrhizal fungus Tuber borchii. A peculiar feature of this phospholipase and of its ortholog from the black truffle Tuber melanosporum is the presence of a 54-amino acid sequence of unknown functional significance, interposed between the signal peptide and the start of the conserved catalytic core of the enzyme. X-ray diffraction analysis of a recombinant TbSP1 form corresponding to the secreted protein previously identified in T. borchii mycelia revealed a structure comprising the five α-helices that form the phospholipase catalytic module but lacking the N-terminal 54 amino acids. This finding led to a series of functional studies that showed that TbSP1, as well as its T. melanosporum ortholog, is a self-processing pro-phospholipase A(2), whose phospholipase activity increases up to 80-fold following autoproteolytic removal of the N-terminal peptide. Proteolytic cleavage occurs within a serine-rich, intrinsically flexible region of TbSP1, does not involve the phospholipase active site, and proceeds via an intermolecular mechanism. Autoproteolytic activation, which also takes place at the surface of nutrient-starved, sPLA(2) overexpressing hyphae, may strengthen and further control the effects of phospholipase up-regulation in response to nutrient deprivation, also in the context of symbiosis establishment and mycorrhiza formation.
View details for DOI 10.1074/jbc.M112.384156
View details for Web of Science ID 000313751400011
View details for PubMedID 23192346
Amicyanin Transfers Electrons from Methylamine Dehydrogenase to Cytochrome c-551i via a Ping-Pong Mechanism, not a Ternary Complex
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2010; 132 (41): 14537-14545
In many biochemical processes, proteins need to bind partners amidst a sea of other molecules. Generally, partner selection is achieved by formation of a single-orientation complex with well-defined, short-range interactions. We describe a protein network that functions effectively in a metabolic electron transfer process but lacks such specific interactions. The soil bacterium Paracoccus denitrificans oxidizes a variety of compounds by channeling electrons into the main respiratory pathway. Upon conversion of methylamine by methylamine dehydrogenase, electrons are transported to the terminal oxidase to reduce molecular oxygen. Steady-state kinetic measurements and NMR experiments demonstrate a remarkable number of possibilities for the electron transfer, involving the cupredoxin amicyanin as well as four c-type cytochromes. The observed interactions appear to be governed exclusively by the electrostatic nature of each of the proteins. It is concluded that Paracoccus provides a pool of cytochromes for efficient electron transfer via weak, ill-defined interactions, in contrast with the view that functional biochemical interactions require well-defined molecular interactions. It is proposed that the lack of requirement for specificity in these interactions might facilitate the integration of new metabolic pathways.
View details for DOI 10.1021/ja205043f
View details for Web of Science ID 000296678200033
View details for PubMedID 21916462
The first crystal structure of a ternary redox protein complex was comprised of the enzyme methylamine dehydrogenase (MADH) and two electron transfer proteins, amicyanin and cytochrome c-551i from Paracoccus denitrificans [Chen et al. Science 1994, 264, 86-90]. The arrangement of the proteins suggested possible electron transfer from the active site of MADH via the amicyanin copper ion to the cytochrome heme iron, although the distance between the metals is large. We studied the interactions between these proteins in solution. A titration followed by NMR spectroscopy shows that amicyanin binds cytochrome c-551i. The interface comprises the hydrophobic and positive patches of amicyanin, not the binding site observed in the ternary complex. NMR experiments further show that amicyanin binds tightly to MADH with an interface that matches the one observed in the crystal structure and that mostly overlaps with the binding site for cytochrome c-551i. Upon addition of cytochrome c-551i, no changes in the NMR spectrum of MADH-bound amicyanin are observed, suggesting that a possible interaction of the cytochrome with the binary complex must be very weak, with a dissociation constant higher than 2 mM. Reconstitution of the entire redox chain in vitro demonstrates that amicyanin can react rapidly with cytochrome c-551i, but that association of amicyanin with MADH inhibits this reaction. It is concluded that electron transfer from MADH to cytochrome c-551i does not involve a ternary complex but occurs via a ping-pong mechanism in which amicyanin uses the same interface for the reactions with MADH and cytochrome c-551i.
View details for DOI 10.1021/ja105498m
View details for Web of Science ID 000283276800048
View details for PubMedID 20873742