New substrates and activity of Phanerochaete chrysosporium Omega glutathione transferases

Omega glutathione transferases (GSTO) constitute a family of proteins with variable distribution throughout living organisms. It is notably expanded in several fungi and particularly in the wood-degrading fungus Phanerochaete chrysosporium, raising questions concerning the function(s) and potential redundancy of these enzymes. Within the fungal families, GSTOs have been poorly studied and their functions remain rather sketchy. In this study, we have used fluorescent compounds as activity reporters to identify putative ligands. Experiments using 5-chloromethylfluorescein diacetate as a tool combined with mass analyses showed that GSTOs are able to cleave ester bonds. Using this property, we developed a specific activity-based profiling method for identifying ligands of PcGSTO3 and PcGSTO4. The results suggest that GSTOs could be involved in the catabolism of toxic compounds like tetralone derivatives. Biochemical investigations demonstrated that these enzymes are able to catalyze deglutathionylation reactions thanks to the presence of a catalytic cysteine residue. To access the physiological function of these enzymes and notably during the wood interaction, recombinant proteins have been immobilized on CNBr Sepharose and challenged with beech wood extracts. Coupled with GC–MS experiments this ligand fishing method allowed to identify terpenes as potential substrates of Omega GST suggesting a physiological role during the wood–fungus interactions.     

Insights into deglutathionylation reactions. Different intermediates in the glutaredoxin and protein disulfide isomerase catalyzed reactions are defined by the gamma-linkage present in glutathione

Glutaredoxins are small proteins with a conserved active site (-CXX(C/S)-) and thioredoxin fold. These thiol disulfide oxidoreductases catalyze disulfide reductions, preferring GSH-mixed disulfides as substrates. We have developed a new real-time fluorescence-based method for measuring the deglutathionylation activity of glutaredoxins using a glutathionylated peptide as a substrate. Mass spectrometric analysis showed that the only intermediate in the reaction is the glutaredoxin-GSH mixed disulfide. This specificity was solely dependent on the unusual gamma-linkage present in glutathione. The deglutathionylation activity of both wild-type Escherichia coli glutaredoxin and the C14S mutant was competitively inhibited by oxidized glutathione, with K(i) values similar to the K(m) values for the glutathionylated peptide substrate, implying that glutaredoxin primarily recognizes the substrate via the glutathione moiety. In addition, wild-type glutaredoxin showed a sigmoidal dependence on GSH concentrations, the activity being significantly decreased at low GSH concentrations. Thus, under oxidative stress conditions, where the ratio of GSH/GSSG is decreased, the activity of glutaredoxin is dramatically reduced, and it will only have significant deglutathionylation activity once the oxidative stress has been removed. Different members of the protein disulfide isomerases (PDI) family showed lower activity levels when compared with glutaredoxins; however, their deglutathionylation activities were comparable with their oxidase activities. Furthermore, in contrast to the glutaredoxin-GSH mixed disulfide intermediate, the only intermediate in the PDI-catalyzed reaction was PDI peptide mixed disulfide.     

Investigations of the catalytic mechanism of thioredoxin glutathione reductase from Schistosoma mansoni

Thioredoxin glutathione reductase from Schistosoma mansoni (SmTGR) catalyzes the reduction of both thioredoxin and glutathione disulfides (GSSG), thus playing a crucial role in maintaining redox homeostasis in the parasite. In line with this role, previous studies have demonstrated that SmTGR is a promising drug target for schistosomiasis. To aid in the development of efficacious drugs that target SmTGR, it is essential to understand the catalytic mechanism of SmTGR. SmTGR is a dimeric flavoprotein in the glutathione reductase family and has a head-to-tail arrangement of its monomers; each subunit has the components of both a thioredoxin reductase (TrxR) domain and a glutaredoxin (Grx) domain. However, the active site of the TrxR domain is composed of residues from both subunits: FAD and a redox-active Cys-154/Cys-159 pair from one subunit and a redox-active Cys-596'/Sec-597' pair from the other; the active site of the Grx domain contains a redox-active Cys-28/Cys-31 pair. Via its Cys-28/Cys-31 dithiol and/or its Cys-596'/Sec-597' thiol-selenolate, SmTGR can catalyze the reduction of a variety of substrates by NADPH. It is presumed that SmTGR catalyzes deglutathionylation reactions via the Cys-28/Cys-31 dithiol. Our anaerobic titration data suggest that reducing equivalents from NADPH can indeed reach the Cys-28/Cys-31 disulfide in the Grx domain to facilitate reductions effected by this cysteine pair. To clarify the specific chemical roles of each redox-active residue with respect to its various reactivities, we generated variants of SmTGR. Cys-28 variants had no Grx deglutathionylation activity, whereas Cys-31 variants retained partial Grx deglutathionylation activity, indicating that the Cys-28 thiolate is the nucleophile initiating deglutathionylation. Lags in the steady-state kinetics, found when wild-type SmTGR was incubated at high concentrations of GSSG, were not present in Grx variants, indicating that this cysteine pair is in some way responsible for the lags. A Sec-597 variant was still able to reduce a variety of substrates, albeit slowly, showing that selenocysteine is important but is not the sole determinant for the broad substrate tolerance of the enzyme. Our data show that Cys-520 and Cys-574 are not likely to be involved in the catalytic mechanism.     

Kinetic and mechanistic characterization and versatile catalytic properties of mammalian glutaredoxin 2: implications for intracellular roles

Glutaredoxin (Grx)-catalyzed deglutathionylation of protein-glutathione mixed disulfides (protein-SSG) serves important roles in redox homeostasis and signal transduction, regulating diverse physiological and pathophysiological events. Mammalian cells have two Grx isoforms: Grx1, localized to the cytosol and mitochondrial intermembrane space, and Grx2, localized primarily to the mitochondrial matrix [Pai, H. V., et al. (2007) Antioxid. Redox Signaling 9, 2027-2033]. The catalytic behavior of Grx1 has been characterized extensively, whereas Grx2 catalysis is less well understood. We observed that human Grx1 and Grx2 exhibit key catalytic similarities, including selectivity for protein-SSG substrates and a nucleophilic, double-displacement, monothiol mechanism exhibiting a strong commitment to catalysis. A key distinction between Grx1- and Grx2-mediated deglutathionylation is decreased catalytic efficiency ( k cat/ K M) of Grx2 for protein deglutathionylation (due primarily to a decreased k cat), reflecting a higher p K a of its catalytic cysteine, as well as a decreased enhancement of nucleophilicity of the second substrate, GSH. As documented previously for hGrx1 [Starke, D. W., et al. (2003) J. Biol. Chem. 278, 14607-14613], hGrx2 catalyzes glutathione-thiyl radical (GS (*)) scavenging, and it also mediates GS transfer (protein S-glutathionylation) reactions, where GS (*) serves as a superior glutathionyl donor substrate for formation of GAPDH-SSG, compared to GSNO and GSSG. In contrast to its lower k cat for deglutathionylation reactions, Grx2 promotes GS-transfer to the model protein substrate GAPDH at rates equivalent to those of Grx1. Estimation of Grx1 and Grx2 concentrations within mitochondria predicts comparable deglutathionylation activities within the mitochondrial subcompartments, suggesting localized regulatory functions for both isozymes.     

Crystal Structure of the Human Ubiquitin-activating Enzyme 5 (UBA5) Bound to ATP: MECHANISTIC INSIGHTS INTO A MINIMALISTIC E1 ENZYME*

E1 ubiquitin-activating enzymes (UBAs) are large multidomain proteins that catalyze formation of a thioester bond between the terminal carboxylate of a ubiquitin or ubiquitin-like modifier (UBL) and a conserved cysteine in an E2 protein, producing reactive ubiquityl units for subsequent ligation to substrate lysines. Two important E1 reaction intermediates have been identified: a ubiquityl-adenylate phosphoester and a ubiquityl-enzyme thioester. However, the mechanism of thioester bond formation and its subsequent transfer to an E2 enzyme remains poorly understood. We have determined the crystal structure of the human UFM1 (ubiquitin-fold modifier 1) E1-activating enzyme UBA5, bound to ATP, revealing a structure that shares similarities with both large canonical E1 enzymes and smaller ancestral E1-like enzymes. In contrast to other E1 active site cysteines, which are in a variably sized domain that is separate and flexible relative to the adenylation domain, the catalytic cysteine of UBA5 (Cys²⁵⁰) is part of the adenylation domain in an α-helical motif. The novel position of the UBA5 catalytic cysteine and conformational changes associated with ATP binding provides insight into the possible mechanisms through which the ubiquityl-enzyme thioester is formed. These studies reveal structural features that further our understanding of the UBA5 enzyme reaction mechanism and provide insight into the evolution of ubiquitin activation.     

A Role for Glutathione Transferase Omega 1 (GSTO1-1) in the Glutathionylation Cycle

The glutathionylation of intracellular protein thiols can protect against irreversible oxidation and can act as a redox switch regulating metabolic pathways. In this study we discovered that the Omega class glutathione transferase GSTO1-1 plays a significant role in the glutathionylation cycle. The catalytic activity of GSTO1-1 was determined in vitro by assaying the deglutathionylation of a synthetic peptide by tryptophan fluorescence quenching and in T47-D epithelial breast cancer cells by both immunoblotting and the direct determination of total glutathionylation. Mutating the active site cysteine residue (Cys-32) ablated the deglutathionylating activity of GSTO1-1. Furthermore, we demonstrate that the expression of GSTO1-1 in T47-D cells that are devoid of endogenous GSTO1-1 resulted in a 50% reduction in total glutathionylation levels. Mass spectrometry and immunoprecipitation identified β-actin as a protein that is specifically deglutathionylated by GSTO1-1 in T47-D cells. In contrast to the deglutathionylation activity, we also found that GSTO1-1 is associated with the rapid glutathionylation of cellular proteins when the cells are exposed to S-nitrosoglutathione. The common A140D genetic polymorphism in GSTO1 was found to have significant effects on the kinetics of both the deglutathionylation and glutathionylation reactions. Genetic variation in GSTO1-1 has been associated with a range of diseases, and the discovery that a frequent GSTO1-1 polymorphism affects glutathionylation cycle reactions reveals a common mechanism where it can act on multiple proteins and pathways.     

Biochemical and Structural Studies of 6-Carboxy-5,6,7,8-tetrahydropterin Synthase Reveal the Molecular Basis of Catalytic Promiscuity within the Tunnel-fold Superfamily

6-Pyruvoyltetrahydropterin synthase (PTPS) homologs in both mammals and bacteria catalyze distinct reactions using the same 7,8-dihydroneopterin triphosphate substrate. The mammalian enzyme converts 7,8-dihydroneopterin triphosphate to 6-pyruvoyltetrahydropterin, whereas the bacterial enzyme catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin. To understand the basis for the differential activities we determined the crystal structure of a bacterial PTPS homolog in the presence and absence of various ligands. Comparison to mammalian structures revealed that although the active sites are nearly structurally identical, the bacterial enzyme houses a His/Asp dyad that is absent from the mammalian protein. Steady state and time-resolved kinetic analysis of the reaction catalyzed by the bacterial homolog revealed that these residues are responsible for the catalytic divergence. This study demonstrates how small variations in the active site can lead to the emergence of new functions in existing protein folds.     

Role of Glutaredoxin1 and Glutathione in Regulating the Activity of the Copper-transporting P-type ATPases,ATP7A and ATP7B

The copper-transporting P-type ATPases (Cu-ATPases), ATP7A and ATP7B, are essential for the regulation of intracellular copper homeostasis. In this report we describe new roles for glutathione (GSH) and glutaredoxin1 (GRX1) in Cu homeostasis through their regulation of Cu-ATPase activity. GRX1 is a thiol oxidoreductase that catalyzes the reversible reduction of GSH-mixed disulfides to their respective sulfhydryls (deglutathionylation). Here, we demonstrated that glutathionylation of the Cu-ATPases and their interaction with GRX1 were affected by alterations in Cu levels. The data support our hypothesis that the Cu-ATPases serve as substrates for Cu-dependent GRX1-mediated deglutathionylation. This in turn liberates the Cu-ATPase cysteinyl thiol groups for Cu binding and transport. GSH depletion experiments led to reversible inhibition of the Cu-ATPases that correlated with effects on intracellular Cu levels and GRX1 activity. Finally, knockdown of GRX1 expression resulted in an increase in intracellular Cu accumulation. Together, these data directly implicate GSH and GRX1 with important new roles in redox regulation of the Cu-ATPases, through modulation of Cu binding by the Cu-ATPase cysteine motifs.     

Methionine sulfoxide reductase: Chemistry,substrate binding,recycling process and oxidase activity

Three classes of methionine sulfoxide reductases are known: MsrA and MsrB which are implicated stereo-selectively in the repair of protein oxidized on their methionine residues; and fRMsr, discovered more recently, which binds and reduces selectively free L-Met-R-O. It is now well established that the chemical mechanism of the reductase step passes through formation of a sulfenic acid intermediate. The oxidized catalytic cysteine can then be recycled by either Trx when a recycling cysteine is operative or a reductant like glutathione in the absence of recycling cysteine which is the case for 30% of the MsrBs. Recently, it was shown that a subclass of MsrAs with two recycling cysteines displays an oxidase activity. This reverse activity needs the accumulation of the sulfenic acid intermediate. The present review focuses on recent insights into the catalytic mechanism of action of the Msrs based on kinetic studies, theoretical chemistry investigations and new structural data. Major attention is placed on how the sulfenic acid intermediate can be formed and the oxidized catalytic cysteine returns back to its reduced form.     

Crystal structure of YnjE from Escherichia coli,a sulfurtransferase with three rhodanese domains

Rhodaneses/sulfurtransferases are ubiquitous enzymes that catalyze the transfer of sulfane sulfur from a donor molecule to a thiophilic acceptor via an active site cysteine that is modified to a persulfide during the reaction. Here, we present the first crystal structure of a triple‐domain rhodanese‐like protein, namely YnjE from Escherichia coli, in two states where its active site cysteine is either unmodified or present as a persulfide. Compared to well‐characterized tandem domain rhodaneses, which are composed of one inactive and one active domain, YnjE contains an extra N‐terminal inactive rhodanese‐like domain. Phylogenetic analysis reveals that YnjE triple‐domain homologs can be found in a variety of other γ‐proteobacteria, in addition, some single‐, tandem‐, four and even six‐domain variants exist. All YnjE rhodaneses are characterized by a highly conserved active site loop (CGTGWR) and evolved independently from other rhodaneses, thus forming their own subfamily. On the basis of structural comparisons with other rhodaneses and kinetic studies, YnjE, which is more similar to thiosulfate:cyanide sulfurtransferases than to 3‐mercaptopyruvate:cyanide sulfurtransferases, has a different substrate specificity that depends not only on the composition of the active site loop with the catalytic cysteine at the first position but also on the surrounding residues. In vitro YnjE can be efficiently persulfurated by the cysteine desulfurase IscS. The catalytic site is located within an elongated cleft, formed by the central and C‐terminal domain and is lined by bulky hydrophobic residues with the catalytic active cysteine largely shielded from the solvent.     

Redox control of caspase-3 activity by thioredoxin and other reduced proteins

Caspases are cysteine proteinases that play a critical role in the execution phase of apoptosis. The active site cysteine residue must be reduced for caspase activity. Thioredoxins are redox proteins that catalyze the reduction of cysteine residues. We have examined the ability of various recombinant human thioredoxins to activate caspase-3. The EC(50) for caspase-3 activation by reduced thioredoxin-1 was 2.5 microM, by reduced glutathione 1.0 mM and by dithiothreitol 3.5 mM. A catalytic site redox-inactive mutant thioredoxin-1 was almost as active as thioredoxin-1 in activating caspase-3. Caspase activation was shown to correlate with the number of reduced cysteine residues in the thioredoxins. Reduced insulin and serum albumin were as effective on a molar basis as thioredoxin-1 in activating caspase-3. Thus, caspase-3 activation is not a specific effect of thioredoxins but is a property shared by other reduced proteins.     

Novel Insights into Eukaryotic γ-Glutamyltranspeptidase 1 from the Crystal Structure of the Glutamate-bound Human Enzyme

The enzyme γ-glutamyltranspeptidase 1 (GGT1) is a conserved member of the N-terminal nucleophile hydrolase family that cleaves the γ-glutamyl bond of glutathione and other γ-glutamyl compounds. In animals, GGT1 is expressed on the surface of the cell and has critical roles in maintaining cysteine levels in the body and regulating intracellular redox status. Expression of GGT1 has been implicated as a potentiator of asthma, cardiovascular disease, and cancer. The rational design of effective inhibitors of human GGT1 (hGGT1) has been delayed by the lack of a reliable structural model. The available crystal structures of several bacterial GGTs have been of limited use due to differences in the catalytic behavior of bacterial and mammalian GGTs. We report the high resolution (1.67 Å) crystal structure of glutamate-bound hGGT1, the first of any eukaryotic GGT. Comparisons of the active site architecture of hGGT1 with those of its bacterial orthologs highlight key differences in the residues responsible for substrate binding, including a bimodal switch in the orientation of the catalytic nucleophile (Thr-381) that is unique to the human enzyme. Compared with several bacterial counterparts, the lid loop in the crystal structure of hGGT1 adopts an open conformation that allows greater access to the active site. The hGGT1 structure also revealed tightly bound chlorides near the catalytic residue that may contribute to catalytic activity. These are absent in the bacterial GGTs. These differences between bacterial and mammalian GGTs and the new structural data will accelerate the development of new therapies for GGT1-dependent diseases.     

A Structural Element within the HUWE1 HECT Domain Modulates Self-ubiquitination and Substrate Ubiquitination Activities

E3 ubiquitin ligases catalyze the final step of ubiquitin conjugation and regulate numerous cellular processes. The HECT class of E3 ubiquitin (Ub) ligases directly transfers Ub from bound E2 enzyme to a myriad of substrates. The catalytic domain of HECT Ub ligases has a bilobal architecture that separates the E2 binding region and catalytic site. An important question regarding HECT domain function is the control of ligase activity and specificity. Here we present a functional analysis of the HECT domain of the E3 ligase HUWE1 based on crystal structures and show that a single N-terminal helix significantly stabilizes the HECT domain. We observe that this element modulates HECT domain activity, as measured by self-ubiquitination induced in the absence of this helix, as distinct from its effects on Ub conjugation of substrate Mcl-1. Such subtle changes to the protein may be at the heart of the vast spectrum of substrate specificities displayed by HECT domain E3 ligases.     

Engineering a new C-terminal tail in the H-site of human glutathione transferase P1-1: structural and functional consequences

We have sought the structural basis for the differing substrate specificities of human glutathione transferase P1-1 (class Pi) and human glutathione transferase A1-1 (class Alpha) by adding an extra helix (helix 9), found in the electrophilic substrate-binding site (H-site) of the human class Alpha enzyme, at the C terminus of the human class Pi enzyme. This class Pi-chimera (CODA) was expressed in Escherichia coli, purified and characterized by kinetic and crystallographic approaches. The presence of the newly engineered tail in the H-site of the human Pi enzyme alters its catalytic properties towards those exhibited by the human Alpha enzyme, as assessed using cumene hydroperoxide (diagnostic for class Alpha enzymes) and ethacrynic acid (diagnostic for class Pi) as co-substrates. There is a change of substrate selectivity in the latter case, as the k(cat)/K(m)(EA) value decreases about 70-fold, compared to that of class Pi. With 1-chloro-2,4-dinitrobenzene as co-substrate there is a loss of catalytic activity to about 2% with respect to that of the Pi enzyme. Crystallographic and kinetic studies of the class Pi-chimera provide important clues to explain these altered catalytic properties. The new helix forms many complimentary interactions with the rest of the protein and re-models the original electrophilic substrate-binding site towards one that is more enclosed, albeit flexible. Of particular note are the interactions between Glu205 of the new tail and the catalytic residues, Tyr7 and Tyr108, and the thiol moiety of glutathione (GSH). These interactions may provide an explanation of the more than one unit increase in the pK(a) value of the GSH thiolate and affect both the turnover number and GSH binding, using 1-chloro-2,4-dinitrobenzene as co-substrate. The data presented are consistent with the engineered tail adopting a highly mobile or disordered state in the apo form of the enzyme.     

Structure and Kinetic Analysis of H2S Production by Human Mercaptopyruvate Sulfurtransferase

Mercaptopyruvate sulfurtransferase (MST) is a source of endogenous H₂S, a gaseous signaling molecule implicated in a wide range of physiological processes. The contribution of MST versus the other two H₂S generators, cystathionine β-synthase and γ-cystathionase, has been difficult to evaluate because many studies on MST have been conducted at high pH and have used varied reaction conditions. In this study, we have expressed, purified, and crystallized human MST in the presence of the substrate 3-mercaptopyruvate (3-MP). The kinetics of H₂S production by MST from 3-MP was studied at pH 7.4 in the presence of various physiological persulfide acceptors: cysteine, dihydrolipoic acid, glutathione, homocysteine, and thioredoxin, and in the presence of cyanide. The crystal structure of MST reveals a mixture of the product complex containing pyruvate and an active site cysteine persulfide (Cys²⁴⁸-SSH) and a nonproductive intermediate in which 3-MP is covalently linked via a disulfide bond to an active site cysteine. The crystal structure analysis allows us to propose a detailed mechanism for MST in which an Asp-His-Ser catalytic triad is positioned to activate the nucleophilic cysteine residue and participate in general acid-base chemistry, whereas our kinetic analysis indicates that thioredoxin is likely to be the major physiological persulfide acceptor for MST.     

Cysteine biosynthetic enzymes are the pieces of a metabolic energy pump

Understanding the mechanisms of free energy transfer in metabolism is fundamental to understanding how the chemical forces that sustain the molecular organization of the cell are distributed. Recent studies of molecular motors (1-3) and ATP-driven proton transport (4-6) describe how chemical potential is transferred at the molecular level. These systems catalyze energy transfer through structural change and appear to be dedicated exclusively to their coupling tasks (7, 8). Here we report the discovery of a new class of energy-transfer system. It is a biosynthetic pump composed of cysteine biosynthesis enzymes, ATP sulfurylase and O-acetylserine sulfhydrylase, each with its own catalytic function and from whose interactions emerge new function: the hydrolysis of ATP. The hydrolysis is kinetically and energetically linked to the chemistry catalyzed by ATP sulfurylase, the first enzyme in the cysteine biosynthetic pathway, in such a way that each molecule of ATP hydrolyzed, each stroke of the pump, produces 1 equivalent of that enzyme's product. These findings integrate cysteine metabolism and broaden our understanding of the ways in which higher order allostery is used to effect free energy transfer.     

Structural and Computational Studies of the Staphylococcus aureus Sortase B-Substrate Complex Reveal a Substrate-stabilized Oxyanion Hole

Sortase cysteine transpeptidases covalently attach proteins to the bacterial cell wall or assemble fiber-like pili that promote bacterial adhesion. Members of this enzyme superfamily are widely distributed in Gram-positive bacteria that frequently utilize multiple sortases to elaborate their peptidoglycan. Sortases catalyze transpeptidation using a conserved active site His-Cys-Arg triad that joins a sorting signal located at the C terminus of their protein substrate to an amino nucleophile located on the cell surface. However, despite extensive study, the catalytic mechanism and molecular basis of substrate recognition remains poorly understood. Here we report the crystal structure of the Staphylococcus aureus sortase B enzyme in a covalent complex with an analog of its NPQTN sorting signal substrate, revealing the structural basis through which it displays the IsdC protein involved in heme-iron scavenging from human hemoglobin. The results of computational modeling, molecular dynamics simulations, and targeted amino acid mutagenesis indicate that the backbone amide of Glu²²⁴ and the side chain of Arg²³³ form an oxyanion hole in sortase B that stabilizes high energy tetrahedral catalytic intermediates. Surprisingly, a highly conserved threonine residue within the bound sorting signal substrate facilitates construction of the oxyanion hole by stabilizing the position of the active site arginine residue via hydrogen bonding. Molecular dynamics simulations and primary sequence conservation suggest that the sorting signal-stabilized oxyanion hole is a universal feature of enzymes within the sortase superfamily.     

Catalytic and structural diversity of the fluazifop-inducible glutathione transferases from Phaseolus vulgaris

Plant glutathione transferases (GSTs) comprise a large family of inducible enzymes that play important roles in stress tolerance and herbicide detoxification. Treatment of Phaseolus vulgaris leaves with the aryloxyphenoxypropionic herbicide fluazifop-p-butyl resulted in induction of GST activities. Three inducible GST isoenzymes were identified and separated by affinity chromatography. Their full-length cDNAs with complete open reading frame were isolated using RACE-RT and information from N-terminal amino acid sequences. Analysis of the cDNA clones showed that the deduced amino acid sequences share high homology with GSTs that belong to phi and tau classes. The three isoenzymes were expressed in E. coli and their substrate specificity was determined towards 20 different substrates. The results showed that the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs) catalyze a broad range of reactions and exhibit quite varied substrate specificity. Molecular modeling and structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of these enzymes. These results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris.     

Crystal Structure and Mutational Analysis of Aminoacylhistidine Dipeptidase from Vibrio alginolyticus Reveal a New Architecture of M20 Metallopeptidases

Aminoacylhistidine dipeptidases (PepD, EC 3.4.13.3) belong to the family of M20 metallopeptidases from the metallopeptidase H clan that catalyze a broad range of dipeptide and tripeptide substrates, including l-carnosine and l-homocarnosine. Homocarnosine has been suggested as a precursor for the neurotransmitter γ-aminobutyric acid (GABA) and may mediate the antiseizure effects of GABAergic therapies. Here, we report the crystal structure of PepD from Vibrio alginolyticus and the results of mutational analysis of substrate-binding residues in the C-terminal as well as substrate specificity of the PepD catalytic domain-alone truncated protein PepD^CAT. The structure of PepD was found to exist as a homodimer, in which each monomer comprises a catalytic domain containing two zinc ions at the active site center for its hydrolytic function and a lid domain utilizing hydrogen bonds between helices to form the dimer interface. Although the PepD is structurally similar to PepV, which exists as a monomer, putative substrate-binding residues reside in different topological regions of the polypeptide chain. In addition, the lid domain of the PepD contains an “extra” domain not observed in related M20 family metallopeptidases with a dimeric structure. Mutational assays confirmed both the putative di-zinc allocations and the architecture of substrate recognition. In addition, the catalytic domain-alone truncated PepD^CAT exhibited substrate specificity to l-homocarnosine compared with that of the wild-type PepD, indicating a potential value in applications of PepD^CAT for GABAergic therapies or neuroprotection.