Characterization of Mycobacterium tuberculosis diaminopimelic acid epimerase: paired cysteine residues are crucial for racemization

Recently, the overproduction of Mycobacterium tuberculosis diaminopimelic acid (DAP) epimerase MtDapF in Escherichia coli using a novel codon alteration cloning strategy and the characterization of the purified enzyme was reported. In the present study, the effect of sulphydryl alkylating agents on the in vitro activity of M. tuberculosis DapF was tested. The complete inhibition of the enzyme by 2-nitro-5-thiocyanatobenzoate, 5,5'-dithio-bis(2-nitrobenzoic acid) and 1,2-benzisothiazolidine-3-one at nanomolar concentrations suggested that these sulphydryl alkylating agents modify functionally significant cysteine residues at or near the active site of the epimerase. Consequently, the authors extended the characterization of MtDapF by studying the role of the two strictly conserved cysteine residues. The putative catalytic residues Cys87 and Cys226 of MtDapF were replaced individually with both serine and alanine. Residual epimerase activity was detected for both the serine replacement mutants C87S and C226S in vitro. Kinetic analyses revealed that, despite a decrease in the K(M) value of the C87S mutant for DAP that presumably indicates an increase in nonproductive substrate binding, the catalytic efficiency of both serine substitution mutants was severely compromised. When either C87 or C226 were substituted with alanine, epimerase activity was not detected emphasizing the importance of both of these cysteine residues in catalysis.     

Dynamics of catalysis revealed from the crystal structures of mutants of diaminopimelate epimerase

Diaminopimelate (DAP) epimerase catalyzes the stereoinversion of ll-DAP to meso-DAP, a precursor of l-lysine and an essential component of the bacterial peptidoglycan. This function is vital to bacteria and the enzyme therefore represents an attractive target for the design of novel anti-bacterials. DAP epimerase belongs to the group of PLP-independent amino acid racemases that function through a rather unusual mechanism involving two cysteines acting in concert as a base (thiolate) and an acid (thiol). We have solved the crystal structures of the apo-forms of DAP epimerase mutants (C73S and C217S) from Haemophilus influenzae at 2.3A and 2.2A resolution, respectively. These structures provide a snapshot of the enzyme in the first step of the catalytic cycle. Comparisons with the structures of the inhibitor-bound form reveal that the enzyme adopts an 'open conformation' in the absence of substrates or inhibitors with the two active site cysteines existing as a thiol-thiolate pair. Substrate binding to the C-terminal domain triggers the closure of the N-terminal domain coupled with tight encapsulation of the ligand, stabilization of the conformation of an active site loop containing Cys73 and expulsion of water molecules with concomitant desolvation of the thiolate base. This structural rearrangement is critical for catalysis.     

Structural and Kinetic Analysis of Free Methionine-R-sulfoxide Reductase from Staphylococcus aureus: CONFORMATIONAL CHANGES DURING CATALYSIS AND IMPLICATIONS FOR THE CATALYTIC AND INHIBITORY MECHANISMS*

Free methionine-R-sulfoxide reductase (fRMsr) reduces free methionine R-sulfoxide back to methionine, but its catalytic mechanism is poorly understood. Here, we have determined the crystal structures of the reduced, substrate-bound, and oxidized forms of fRMsr from Staphylococcus aureus. Our structural and biochemical analyses suggest the catalytic mechanism of fRMsr in which Cys¹⁰² functions as the catalytic residue and Cys⁶⁸ as the resolving Cys that forms a disulfide bond with Cys¹⁰². Cys⁷⁸, previously thought to be a catalytic Cys, is a non-essential residue for catalytic function. Additionally, our structures provide insights into the enzyme-substrate interaction and the role of active site residues in substrate binding. Structural comparison reveals that conformational changes occur in the active site during catalysis, particularly in the loop of residues 97–106 containing the catalytic Cys¹⁰². We have also crystallized a complex between fRMsr and isopropyl alcohol, which acts as a competitive inhibitor for the enzyme. This isopropyl alcohol-bound structure helps us to understand the inhibitory mechanism of fRMsr. Our structural and enzymatic analyses suggest that a branched methyl group in alcohol seems important for competitive inhibition of the fRMsr due to its ability to bind to the active site.     

Mechanism of Thiosulfate Oxidation in the SoxA Family of Cysteine-ligated Cytochromes

Thiosulfate dehydrogenase (TsdA) catalyzes the oxidation of two thiosulfate molecules to form tetrathionate and is predicted to use an unusual cysteine-ligated heme as the catalytic cofactor. We have determined the structure of Allochromatium vinosum TsdA to a resolution of 1.3 Å. This structure confirms the active site heme ligation, identifies a thiosulfate binding site within the active site cavity, and reveals an electron transfer route from the catalytic heme, through a second heme group to the external electron acceptor. We provide multiple lines of evidence that the catalytic reaction proceeds through the intermediate formation of a S-thiosulfonate derivative of the heme cysteine ligand: the cysteine is reactive and is accessible to electrophilic attack; cysteine S-thiosulfonate is formed by the addition of thiosulfate or following the reverse reaction with tetrathionate; the S-thiosulfonate modification is removed through catalysis; and alkylating the cysteine blocks activity. Active site amino acid residues required for catalysis were identified by mutagenesis and are inferred to also play a role in stabilizing the S-thiosulfonate intermediate. The enzyme SoxAX, which catalyzes the first step in the bacterial Sox thiosulfate oxidation pathway, is homologous to TsdA and can be inferred to use a related catalytic mechanism.     

Chemical mechanism of Haemophilus influenzae diaminopimelate epimerase

Seven unique enzymatic steps lead to the biosynthesis of L-lysine from L-aspartate semialdehyde and pyruvate in bacteria. The immediate precursor to L-lysine is D,L-diaminopimelate, a diamino acid which is incorporated into the pentapeptide of the Gram-negative peptidoglycan moiety. D,L-Diaminopimelate is generated from the corresponding L,L-isomer by the dapF-encoded epimerase. Diaminopimelate epimerase is a representative of the pyridoxal phosphate-independent amino acid racemases, for which substantial evidence exists supporting the role of two cysteine residues as the catalytic acid and base. The pH dependencies of the maximum velocities in the L,L --> D,L and D,L --> L,L direction depend on a single catalytic group exhibiting pK values of 7.0 and 6.1, respectively, which must be unprotonated for activity. The pH dependencies of the V/K values in both directions depend on the ionization of two groups, one exhibiting a pK value of 6.7 which must be unprotonated and one exhibiting a pK value of 8.5 which must be protonated. Primary kinetic isotope effects on V and V/K are unequal, with D(V/K) being larger than DV in both the forward and reverse directions. Solvent kinetic isotope effects in both directions are inverse on V/K, but normal on V. Both of these isotopic observations support a model in which proton isomerization after catalysis and substrate dissociation is kinetically significant. A single solvent "overshoot" is observed when L, L-diaminopimelate is incubated with enzyme in D2O; however, an unprecedented double overshoot is observed when D,L-diaminopimelate is incubated with enzyme in D2O. A model has been developed which allows these two overshoots to be simulated. A chemical mechanism is proposed invoking the function of two cysteine residues, Cys73 and Cys217, observed in the recently determined three-dimensional structure of this enzyme [Cirilli, M., et al. (1998) Biochemistry 37, 16452-16458], as the acid and base in the mechanism.     

Redox, mutagenic and structural studies of the glutaredoxin/arsenate reductase couple from the cyanobacterium Synechocystis sp. PCC 6803

The arsenate reductase from the cyanobacterium Synechocystis sp. PCC 6803 has been characterized in terms of the redox properties of its cysteine residues and their role in the reaction catalyzed by the enzyme. Of the five cysteines present in the enzyme, two (Cys13 and Cys35) have been shown not to be required for catalysis, while Cys8, Cys80 and Cys82 have been shown to be essential. The as-isolated enzyme contains a single disulfide, formed between Cys80 and Cys82, with an oxidation-reduction midpoint potential (E_m) value of − 165 mV at pH 7.0. It has been shown that Cys15 is the only one of the four cysteines present in Synechocystis sp. PCC 6803 glutaredoxin A required for its ability to serve as an electron donor to arsenate reductase, while the other three cysteines (Cys18, Cys36 and Cys70) play no role. Glutaredoxin A has been shown to contain a single redox-active disulfide/dithiol couple, with a two-electron, E_m value of − 220 mV at pH 7.0. One cysteine in this disulfide/dithiol couple has been shown to undergo glutathionylation. An X-ray crystal structure, at 1.8 Å resolution, has been obtained for glutaredoxin A. The probable orientations of arsenate reductase disulfide bonds present in the resting enzyme and in a likely reaction intermediate of the enzyme have been examined by in silico modeling, as has the surface environment of arsenate reductase in the vicinity of Cys8, the likely site for the initial reaction between arsenate and the enzyme.     

Statistical coupling analysis of aspartic proteinases based on crystal structures of the Trichoderma reesei enzyme and its complex with pepstatin A

The crystal structures of an aspartic proteinase from Trichoderma reesei (TrAsP) and of its complex with a competitive inhibitor, pepstatin A, were solved and refined to crystallographic R-factors of 17.9% (R_free = 21.2%) at 1.70 Å resolution and 15.8% (R_free = 19.2%) at 1.85 Å resolution, respectively. The three-dimensional structure of TrAsP is similar to structures of other members of the pepsin-like family of aspartic proteinases. Each molecule is folded in a predominantly β-sheet bilobal structure with the N-terminal and C-terminal domains of about the same size. Structural comparison of the native structure and the TrAsP-pepstatin complex reveals that the enzyme undergoes an induced-fit, rigid-body movement upon inhibitor binding, with the N-terminal and C-terminal lobes tightly enclosing the inhibitor. Upon recognition and binding of pepstatin A, amino acid residues of the enzyme active site form a number of short hydrogen bonds to the inhibitor that may play an important role in the mechanism of catalysis and inhibition. The structures of TrAsP were used as a template for performing statistical coupling analysis of the aspartic protease family. This approach permitted, for the first time, the identification of a network of structurally linked residues putatively mediating conformational changes relevant to the function of this family of enzymes. Statistical coupling analysis reveals coevolved continuous clusters of amino acid residues that extend from the active site into the hydrophobic cores of each of the two domains and include amino acid residues from the flap regions, highlighting the importance of these parts of the protein for its enzymatic activity.     

Dimerization of Bacterial Diaminopimelate Epimerase Is Essential for Catalysis

Diaminopimelate (DAP) epimerase is involved in the biosynthesis of meso-DAP and lysine, which are important precursors for the synthesis of peptidoglycan, housekeeping proteins, and virulence factors in bacteria. Accordingly, DAP epimerase is a promising antimicrobial target. Previous studies report that DAP epimerase exists as a monomeric enzyme. However, we show using analytical ultracentrifugation, X-ray crystallography, and enzyme kinetic analyses that DAP epimerase from Escherichia coli exists as a functional dimer in solution and the crystal state. Furthermore, the 2.0-Å X-ray crystal structure of the E. coli DAP epimerase dimer shows for the first time that the enzyme exists in an open, active conformation. The importance of dimerization was subsequently probed by using site-directed mutagenesis to generate a monomeric mutant (Y268A). Our studies show that Y268A is catalytically inactive, thus demonstrating that dimerization of DAP epimerase is essential for catalysis. Molecular dynamics simulations indicate that the DAP epimerase monomer is inherently more flexible than the dimer, suggesting that dimerization optimizes protein dynamics to support function. Our findings offer insight into the development of novel antimicrobial agents targeting the dimeric antibiotic target DAP epimerase.     

Cysteine endoprotease activity of human ribosomal protein S4 is entirely due to the C-terminal domain,and is consistent with Michaelis–Menten mechanism

Background

It is known that tandem domains of enzymes can carry out catalysis independently or by collaboration. In the case of cysteine proteases, domain sequestration abolishes catalysis because the active site residues are distributed in both domains. The validity of this argument is tested here by using isolated human ribosomal protein S4, which has been recently identified as an unorthodox cysteine protease.

Methods

Cleavage of the peptide substrate Z-FR↓-AMC catalyzed by recombinant C-terminal domain of human S4 (CHS4) is studied by fluorescence-monitored steady-state and stopped-flow kinetic methods. Proteolysis and autoproteolysis were analyzed by electrophoresis.

Results

The CHS4 domain comprised of sequence residues 116–263 has been cloned and ovreexpressed in Escherichia coli. The purified domain is enzymatically active. Barring minor differences, steady-state kinetic parameters for catalysis by CHS4 are very similar to those for full-length human S4. Further, stopped-flow transient kinetics of pre-steady-state substrate binding shows that the catalytic mechanism for both full-length S4 and CHS4 obeys the Michaelis–Menten model adequately. Consideration of the evolutionary domain organization of the S4e family of ribosomal proteins indicates that the central domain (residues 94–170) within CHS4 is indispensable.

Conclusion

The C-terminal domain can carry out catalysis independently and as efficiently as the full-length human S4 does.

Significance

Localization of the enzyme function in the C-terminal domain of human S4 provides the only example of a cysteine endoprotease where substrate-mediated intramolecular domain interaction is irrelevant for catalytic activity.     

A new robust kinetic assay for DAP epimerase activity

DAP epimerase is the penultimate enzyme in the lysine biosynthesis pathway. The most versatile assay for DAP epimerase catalytic activity employs a coupled DAP epimerase–DAP dehydrogenase enzyme system with a commercial mixture of DAP isomers as substrate. DAP dehydrogenase converts meso-DAP to THDP with concomitant reduction of NADP⁺ to NADPH. We show that at high concentrations, accumulation of NADPH results in inhibition of DAPDH, resulting in spurious kinetic data. A new assay has been developed employing DAP decarboxylase that allows the reliable characterisation of DAP epimerase enzyme kinetics.     

Crystal Structure and Putative Mechanism of 3-Methylitaconate-Δ-isomerase from Eubacterium barkeri

3-Methylitaconate-Δ-isomerase (Mii) participates in the nicotinate fermentation pathway of the anaerobic soil bacterium Eubacterium barkeri (order Clostridiales) by catalyzing the reversible conversion of (R)-3-methylitaconate (2-methylene-3-methylsuccinate) to 2,3-dimethylmaleate. The enzyme is also able to catalyze the isomerization of itaconate (methylenesuccinate) to citraconate (methylmaleate) with ca 10-fold higher K_m but > 1000-fold lower k_cat. The gene mii from E. barkeri was cloned and expressed in Escherichia coli. The protein produced with a C-terminal Strep-tag exhibited the same specific activity as the wild-type enzyme. The crystal structure of Mii from E. barkeri has been solved at a resolution of 2.70 Å. The asymmetric unit of the P2₁2₁2₁ unit cell with parameters a = 53.1 Å, b = 142.3 Å, and c = 228.4 Å contains four molecules of Mii. The enzyme belongs to a group of isomerases with a common structural feature, the so-called diaminopimelate epimerase fold. The monomer of 380 amino acid residues has two topologically similar domains exhibiting an α/β-fold. The active site is situated in a cleft between these domains. The four Mii molecules are arranged as a tetramer with 222 symmetry for the N-terminal domains. The C-terminal domains have different relative positions with respect to the N-terminal domains resulting in a closed conformation for molecule A and two distinct open conformations for molecules B and D. The C-terminal domain of molecule C is disordered. The Mii active site contains the putative catalytic residues Lys62 and Cys96, for which mechanistic roles are proposed based on a docking experiment of the Mii substrate complex. The active sites of Mii and the closely related PrpF, most likely a methylaconitate Δ-isomerase, have been compared. The overall architecture including the active-site Lys62, Cys96, His300, and Ser17 (Mii numbering) is similar. This positioning of (R)-3-methylitaconate allows Cys96 (as thiolate) to deprotonate C-3 and (as thiol) to donate a proton to the methylene carbon atom of the resulting allylic carbanion. Interestingly, the active site of isopentenyl diphosphate isomerase type I also contains a cysteine that cooperates with glutamate rather than lysine. It has been proposed that the initial step in this enzyme is a protonation generating a tertiary carbocation intermediate.     

Functional and structural characterization of a thiol peroxidase from Mycobacterium tuberculosis

A thiol peroxidase (Tpx) from Mycobacterium tuberculosis was functionally analyzed. The enzyme shows NADPH-linked peroxidase activity using a thioredoxin-thioredoxin reductase system as electron donor, and anti-oxidant activity in a thiol-dependent metal-catalyzed oxidation system. It reduces H2O2, t-butyl hydroperoxide, and cumene hydroperoxide, and is inhibited by sulfhydryl reagents. Mutational studies revealed that the peroxidatic (Cys60) and resolving (Cys93) cysteine residues are critical amino acids for catalytic activity. The X-ray structure determined to a resolution of 1.75 A shows a thioredoxin fold similar to that of other peroxiredoxin family members. Superposition with structural homologues in oxidized and reduced forms indicates that the M. tuberculosis Tpx is a member of the atypical two-Cys peroxiredoxin family. In addition, the short distance that separates the Calpha atoms of Cys60 and Cys93 and the location of these cysteine residues in unstructured regions may indicate that the M. tuberculosis enzyme is oxidized, though the side-chain of Cys60 is poorly visible. It is solely in the reduced Streptococcus pneumoniae Tpx structure that both residues are part of two distinct helical segments. The M. tuberculosis Tpx is dimeric both in solution and in the crystal structure. Amino acid residues from both monomers delineate the active site pocket.     

Crystal structure of aspartate racemase from Pyrococcus horikoshii OT3 and its implications for molecular mechanism of PLP-independent racemization

There exists a d-enantiomer of aspartic acid in lactic acid bacteria and several hyperthermophilic archaea, which is biosynthesized from the l-enantiomer by aspartate racemase. Aspartate racemase is a representative pyridoxal 5'-phosphate (PLP)-independent amino acid racemase. The "two-base" catalytic mechanism has been proposed for this type of racemase, in which a pair of cysteine residues are utilized as the conjugated catalytic acid and base. We have determined the three-dimensional structure of aspartate racemase from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 at 1.9 A resolution by X-ray crystallography and refined it to a crystallographic R factor of 19.4% (R(free) of 22.2%). This is the first structure reported for aspartate racemase, indeed for any amino acid racemase from archaea. The crystal structure revealed that this enzyme forms a stable dimeric structure with a strong three-layered inter-subunit interaction, and that its subunit consists of two structurally homologous alpha/beta domains, each containing a four-stranded parallel beta-sheet flanked by six alpha-helices. Two strictly conserved cysteine residues (Cys82 and Cys194), which have been shown biochemically to act as catalytic acid and base, are located on both sides of a cleft between the two domains. The spatial arrangement of these two cysteine residues supports the "two-base" mechanism but disproves the previous hypothesis that the active site of aspartate racemase is located at the dimeric interface. The structure revealed a unique pseudo mirror-symmetry in the spatial arrangement of the residues around the active site, which may explain the molecular recognition mechanism of the mirror-symmetric aspartate enantiomers by the non-mirror-symmetric aspartate racemase.     

Structure of the Mature Streptococcal Cysteine Protease Exotoxin mSpeB in Its Active Dimeric Form

Invasive infections of Streptococcus pyogenes are dependent on the cysteine protease streptococcal pyrogenic exotoxin B. Previous structures of the enzyme have not disclosed the proper active-site configuration. Here, the crystal structure of the mature enzyme is presented to 1.55 Å, disclosing a homodimer. A serine from one subunit inserts into the active site of the other to donate to the oxyanion hole and coordinates the ligand proximal to the active-site cysteine. Dimerization is unique to the mature form and is clearly a prerequisite for catalysis. The present structure supports a tripartite switch system that is triggered upon dimerization and substrate binding: (1) liberation of the active-site histidine from an inactive configuration, (2) relocation of residues blocking the substrate binding pockets and (3) repositioning of two active-site tryptophans to settle in the active configuration. Based on the present structure, the active site of clan CA cysteine proteases is expanded and a detailed mechanism of the deacylation mechanism is proposed. The results may have applications for the development of protease inhibitors specific to bacterial cysteine proteases.     

Crystal Structure of Metal-Dependent Allantoinase from Escherichia coli

Allantoinase acts as a key enzyme for the biogenesis and degradation of ureides by catalyzing the conversion of (S)-allantoin into allantoate, the final step in the ureide pathway. Despite limited sequence similarity, biochemical studies of the enzyme suggested that allantoinase belongs to the amidohydrolase family. In this study, the crystal structure of allantoinase from Escherichia coli was determined at 2.1 Å resolution. The enzyme consists of a homotetramer in which each monomer contains two domains: a pseudo-triosephosphate-isomerase barrel and a β-sheet. Analogous to other enzymes in the amidohydrolase family, allantoinase retains a binuclear metal center in the active site, embedded within the barrel fold. Structural analyses demonstrated that the metal ions in the active site ligate one hydroxide and six residues that are conserved among allantoinases from other organisms. Functional analyses showed that the presence of zinc in the metal center is essential for catalysis and enantioselectivity of substrate. Both the metal center and active site residues Asn94 and Ser317 play crucial roles in dictating enzyme activity. These structural and functional features are distinctively different from those of the metal-independent allantoinase, which was very recently identified.     

Dissecting cobamide diversity through structural and functional analyses of the base-activating CobT enzyme of Salmonella enterica

Background

Cobamide diversity arises from the nature of the nucleotide base. Nicotinate mononucleotide (NaMN):base phosphoribosyltransferases (CobT) synthesize α-linked riboside monophosphates from diverse nucleotide base substrates (e.g., benzimidazoles, purines, phenolics) that are incorporated into cobamides.

Methods

Structural investigations of two members of the CobT family of enzymes in complex with various substrate bases as well as in vivo and vitro activity analyses of enzyme variants were performed to elucidate the roles of key amino acid residues important for substrate recognition.

Results

Results of in vitro and in vivo studies of active-site variants of the Salmonella enterica CobT (SeCobT) enzyme suggest that a catalytic base may not be required for catalysis. This idea is supported by the analyses of crystal structures that show that two glutamate residues function primarily to maintain an active conformation of the enzyme. In light of these findings, we propose that proper positioning of the substrates in the active site triggers the attack at the C1 ribose of NaMN.

Conclusion

Whether or not a catalytic base is needed for function is discussed within the framework of the in vitro analysis of the enzyme activity. Additionally, structure-guided site-directed mutagenesis of SeCobT broadened its substrate specificity to include phenolic bases, revealing likely evolutionary changes needed to increase cobamide diversity, and further supporting the proposed mechanism for the phosphoribosylation of phenolic substrates.

General Significance

Results of this study uncover key residues in the CobT enzyme that contribute to the diversity of cobamides in nature.     

Crystal and molecular structures of native and CTP-liganded aspartate carbamoyltransferase from Escherichia coli

Atomic models representing the electron density of two crystalline forms of aspartate carbamoyltransferase from Escherichia coli are reported here. The unliganded form (R32 crystal symmetry) and the CTP-liganded form (P321 crystal symmetry) have been refined independently at resolutions of 3.0 å and 2.8 Å, respectively, each to a crystallographic R-factor of 27%. The molecular models include at least 95% of the theoretical number of atoms for the aspartate Carbamoyltransferase molecule based on chemical sequence information. We provide details of the refinement process for the two structures, and an evaluation of the accuracy of the molecular models.For the most part, the regulatory and catalytic chains of the unliganded enzyme and the CTP-liganded form are in similar conformations. Large conformational differences in the CTP and native forms exist, however, specifically in the region of CTP binding to the regulatory chain. In addition, a segment of ten amino acid residues, which includes Lys83 and Lys84 of the catalytic chain, is disordered in the CTP-liganded form, in contrast to the native structure, where the same residues have refined well into density.Each catalytic monomer of aspartate carbamoyltransferase is in contact with three catalytic chains and two regulatory monomers. Each regulatory monomer borders on one other regulatory chain and two catalytic chains. The catalytic trimera are in contact in the hexamer; residues important to homotropic effects and catalysis (Tyr165 and Tyr232) are integral parts of the interface. We present a thorough survey of interface regions, cataloging polar interactions between sidechains throughout the molecule.We discuss, in context with the present structures, the chemical modifications and mutations of the enzyme. Highlighted specifically are Cys47, Tyr165 and Tyr232, Lys83, Lys84, Trp209 and Trp279 and Gly128, residues of demonstrated importance to the catalytic of regulatory function or aspartate carbamoyltransferase. The spatial arrangement of “active site” residues argues for a catalytic pocket shared between two monomers within catalytic subunit.     

Vibrio cholerae LMWPTP-2 display unique surface charge and grooves around the active site: Indicative of distinctive substrate specificity and scope to design specific inhibitor

Low molecular weight protein tyrosine phosphatases (LMWPTPs) are ubiquitously found as small cytoplasmic enzymes which act on phospho-tyrosine containing proteins that are engaged in various cellular functions. Vibrio cholerae O395 contains two LMWPTPs having widely different sequence. Phylogenetic analysis based on a non redundant set of 124 LMWPTP sequences, designate that LMWPTP-2 from Vibrio choleraeO395 (VcLMWPTP-2) is a single taxon. We have determined the crystal structure of VcLMWPTP-2 at 2.6 Å with MOPS bound in the active site. Tertiary structure analysis indicates that VcLMWPTP-2 forms dimer. Studies in solution state also confirm exclusive presence of a dimeric form. Kinetic studies demonstrate that VcLMWPTP-2 dimer is catalytically active while inactivation through oligomerisation was reported as one of the regulatory mechanism in case of mammalian LMWPTP viz., Bos taurus LMWPTP, BPTP. Kinetic studies using p-nitrophenyl phosphate (p-NPP) as a substrate demonstrate active participation of both the P-loop cysteine in catalysis. Vicinal Cys17, in addition plays a role of protecting the catalytic Cys12 under oxidative stress. Structural analysis and MD simulations allowed us to propose the role of several conserved residues around the active site. Distribution of surface charges and grooves around the active site delineates unique features of VcLMWPTP-2 which could be utilized to design specific inhibitor.     

Crystallographic and Functional Characterization of the Fluorodifen-inducible Glutathione Transferase from Glycine max Reveals an Active Site Topography Suited for Diphenylether Herbicides and a Novel L-site

Glutathione transferases (GSTs) from the tau class (GSTU) are unique to plants and have important roles in stress tolerance and the detoxification of herbicides in crops and weeds. A fluorodifen-induced GST isoezyme (GmGSTU4-4) belonging to the tau class was purified from Glycine max by affinity chromatography. This isoenzyme was cloned and expressed in Escherichia coli, and its structural and catalytic properties were investigated. The structure of GmGSTU4-4 was determined at 1.75 Å resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). The enzyme adopts the canonical GST fold but with a number of functionally important differences. Compared with other plant GSTs, the three-dimensional structure of GmGSTU4-4 primarily shows structural differences in the hydrphobic substrate binding site, the linker segment and the C-terminal region. The X-ray structure identifies key amino acid residues in the hydrophobic binding site (H-site) and provides insights into the substrate specificity and catalytic mechanism of the enzyme. The isoenzyme was highly active in conjugating the diphenylether herbicide fluorodifen. A possible reaction pathway involving the conjugation of glutathione with fluorodifen is described based on site-directed mutagenesis and molecular modeling studies. A serine residue (Ser13) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione and enhance its nucleophilicity. Tyr107 and Arg111 present in the active site are important structural moieties that modulate the catalytic efficiency and specificity of the enzyme, and participate in k_cat regulation by affecting the rate-limiting step of the catalytic reaction. A hitherto undescribed ligand-binding site (L-site) located in a surface pocket of the enzyme was also found. This site is formed by conserved residues, suggesting it may have an important functional role in the transfer and delivery of bound ligands, presumably to specific protein receptors.     

Understanding the Functional Roles of Amino Acid Residues in Enzyme Catalysis

The MACiE database contains 223 distinct step-wise enzyme reaction mechanisms and holds representatives from each EC sub-subclass where there is a crystal structure and sufficient evidence in the literature to support a mechanism. Each catalytic step of every reaction sequence in MACiE is fully annotated so that it includes the function of the catalytic residues involved in the reaction and the mechanism by which substrates are transformed into products. Using MACiE as a knowledge base, we have seen that the top 10 most catalytic residues are histidine, aspartate, glutamate, lysine, cysteine, arginine, serine, threonine, tyrosine and tryptophan. Of these only seven (cysteine, histidine, aspartate, lysine, serine, threonine and tyrosine) dominate catalysis and provide essentially five functional roles that are essential. Stabilisation is the most common and essential role for all classes of enzyme, followed by general acid/base (proton acceptor and proton donor) functionality, with nucleophilic addition following closely behind (nucleophile and nucleofuge). We investigated the occurrence of these residues in MACiE and the Catalytic Site Atlas and found that, as expected, certain residue types are associated with each functional role, with some residue types able to perform diverse roles. In addition, it was seen that different EC classes of enzyme have a tendency to employ different residues for catalysis. Further, we show that whilst the differences between EC classes in catalytic residue composition are not immediately obvious from the general classes of Ingold mechanisms, there is some weak correlation between the mechanisms involved in a given EC class and the functions that the catalytic amino acid residues are performing. The analysis presented here provides a valuable insight into the functional roles of catalytic amino acid residues, which may have applications in many aspects of enzymology, from the design of novel enzymes to the prediction and validation of enzyme reaction mechanisms.