Probing the role of threonine and serine residues of E. coli asparaginase II by site-specific mutagenesis.

Site-specific mutagenesis has been used to probe amino acid residues proposed to be critical in catalysis by Escherichia coli asparaginase II. Thr12 is conserved in all known asparaginases. The catalytic constant of a T12A mutant towards L-aspartic acid beta-hydroxamate was reduced to 0.04% of wild type activity, while its Km and stability against urea denaturation were unchanged. The mutant enzyme T12S exhibited almost normal activity but altered substrate specificity. Replacement of Thr119 with Ala led to a 90% decrease of activity without markedly affecting substrate binding. The mutant enzyme S122A showed normal catalytic function but impaired stability in urea solutions. These data indicate that the hydroxyl group of Thr12 is directly involved in catalysis, probably by favorably interacting with a transition state or intermediate. By contrast, Thr119 and Ser122, both putative target sites of the inactivator DONV, are functionally less important.     

唾液乳杆菌BSH1及其突变体的底物特异性

为了解析胆盐水解酶催化中心中关键氨基酸位点与其底物特异性的关系,以大肠杆菌pET-20b(+)表达系统为分子改造平台,采用理性设计,结合氨基酸定点突变的方法,成功构建了唾液乳杆菌Lactobacillus salivarius胆盐水解酶BSH1的7种突变体。通过对比L.salivarius BSH1及其突变体对6种结合胆盐的底物特异性表明,7种突变体对不同的结合胆盐的水解活性有所改变。结果说明,Cys2和Thr264分别是BSH1催化TCA和GCA的关键残基,且对酶的催化活性的保持具有关键作用。其中,高保守性的氨基酸位点Cys2不是BSH1唯一的活性位点,而其他突变的氨基酸位点可能作为BSH1的结合位点参与了底物的结合,也可能影响了底物进入BSH1活性中心的通道或底物结合口袋的体积与形状,进而影响了BSH1对不同结合胆盐的水解活性。     

Mutant subtilisin E with enhanced protease activity obtained by site-directed mutagenesis

The specific activity of subtilisin E, an alkaline serine protease of Bacillus subtilis, was substantially increased by optimizing the amino acid residue at position 31 (Ile in the wild-type enzyme) in the vicinity of the catalytic triad of the enzyme. Eight uncharged amino acids (Cys, Ser, Thr, Gly, Ala, Val, Leu, and Phe) were introduced at this site, which is next to catalytic Asp32, using site-directed mutagenesis. Mutant enzymes were expressed in Escherichia coli and were prepared from the periplasmic space. Only the Val and Leu substitutions gave active enzyme, and the Leu31 mutant was found to have a greatly increased activity compared to the wild-type enzyme. The other six mutant enzymes showed a marked decrease in activity. This result indicates that a branched-chain amino acid at position 31 is essential for the expression of subtilisin activity and that the level of the activity depends on side chain structure. The purified Leu31 mutant enzyme was analyzed with respect to substrate specificity, heat stability, and optimal temperature. It was found that the Leu31 replacement caused a prominent 2-6-fold increase in catalytic efficiency (kcat/Km) due to a larger kcat for peptide substrates.     

Mutation of Thr466 in SHP2 abolishes its phosphatase activity, but provides a new substrate-trapping mutant

Most classical phosphotyrosyl phosphatases (PTPs), including the Src homology phosphotyrosyl phosphatase 2 (SHP2) possess a Thr or a Ser residue immediately C-terminal to the invariant Arg in the active site consensus motif (H/V-C-X5-R-S/T), also known as the "signature motif". SHP2 has a Thr (Thr466) at this position, but its importance in catalysis has not been investigated. By employing site-directed mutagenesis, phosphatase assays and substrate-trapping studies, we demonstrate that Thr466 is critical for the catalytic activity of SHP2. Its mutation to Ala abolishes phosphatase activity, but provides a new substrate-trapping mutant. We further show that the nucleophilic Cys459 is not involved in substrate trapping by Thr466Ala-SHP2 (T/A-SHP2). Mutation of Thr466 does not cause significant structural changes in the active site as revealed by the trapping of the epidermal growth factor receptor (EGFR), the physiological substrate of SHP2, and by orthovanadate competition experiments. Based on these results and previous other works, we propose that the role of Thr466 in the catalytic process of SHP2 could be stabilizing the sulfhydryl group of Cys459 in its reduced state, a state that enables nucleophilic attack on the phosphate moiety of the substrate. The T/A-SHP2 harbors a single mutation and specifically interacts with the EGFR. Since the nucleophilic Cys459 and the proton donor Asp425 are intact in the T/A-SAHP2, it offers an excellent starting material for solving the structure of SHP2 in complex with its physiological substrate.     

Systematic exploration of active site mutations on human deoxycytidine kinase substrate specificity

Human deoxycytidine kinase (dCK) is responsible for the phosphorylation of a number of clinically important nucleoside analogue prodrugs in addition to its natural substrates, 2'-deoxycytidine, 2'-deoxyguanosine, and 2'-deoxyadenosine. To improve the low catalytic activity and tailor the substrate specificity of dCK, we have constructed libraries of mutant enzymes and tested them for thymidine kinase (tk) activity. Random mutagenesis was employed to probe for amino acid positions with an impact on substrate specificity throughout the entire enzyme structure, identifying positions Arg104 and Asp133 in the active site as key residues for substrate specificity. Kinetic analysis indicates that Arg104Gln/Asp133Gly creates a "generalist" kinase with broader specificity and elevated turnover for natural and prodrug substrates. In contrast, the substitutions of Arg104Met/Asp133Thr, obtained via site-saturation mutagenesis, yielded a mutant with reversed substrate specificity, elevating the specific constant for thymidine phosphorylation by over 1000-fold while eliminating activity for dC, dA, and dG under physiological conditions. The results illuminate the key contributions of these two amino acid positions to enzyme function by demonstrating their ability to moderate substrate specificity.     

Subunit interface of triosephosphate isomerase: site-directed mutagenesis and characterization of the altered enzyme

We have replaced asparagine residues at the subunit interface of yeast triosephosphate isomerase (TIM) using site-directed mutagenesis in order to elucidate the effects of substitutions on the catalytic activity and conformational stability of the enzyme. The mutant proteins were expressed in a strain of Escherichia coli lacking the bacterial isomerase and purified by ion-exchange and immunoadsorption chromatography. Single replacements of Asn-78 by either Thr or Ile residues had little effect on the enzyme's catalytic efficiency, while the single replacement Asn-78----Asp-78 and the double replacement Asn-14/Asn-78----Thr-14/Ile-78 appreciably lowered kcat for the substrate D-glyceraldehyde 3-phosphate. The isoelectric point of the mutant Asn-78----Asp-78 was equivalent to that of wild-type yeast TIM that had undergone a single, heat-induced deamidation, and this mutant enzyme was less resistant than wild-type TIM to denaturation and inactivation caused by elevated temperature, denaturants, tetrabutylammonium bromide, alkaline pH, and proteases.     

A cold-adapted protease engineered by experimental evolution system.

A new cold-adapted protease subtilisin BPN' mutant, termed m-51, was successfully isolated by use of an evolutionary program consisting of two-step in vitro random mutagenesis, which we developed for the screening of mutant subtilisins with increased activity at low temperature. The m-51 mutant showed 70% higher catalytic efficiency, expressed by the k(cat)/K(m) value, than the wild-type at 10 degrees C against N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as a synthetic substrate. This cold-adaptation was achieved mainly by the increase in the k(cat) value in a temperature-dependent manner. Genetic analysis revealed that m-51 had three mutations, Ala-->Thr at position -31 (A-31T) in the prodomain, Ala-->Val at position 88 (A88V), and Ala-->Thr at position 98 (A98T). From kinetic parameters of the purified mutant enzymes, it was found that the A98T mutation led to 30% activity increase, which was enhanced up to 70% by the accompanying neutral mutation A88V. The A-31T mutation severely constrained the autoprocessing-mediated maturation of the pro-subtilisin in the Escherichia coli expression system, thus probably causing an activity-non-detectable mutation in the first step of mutagenesis. No distinct change was observed in the thermal stability of any mutant or in the substrate specificity for m-51. In the molecular models of the two single mutants (A88V and A98T), relatively large displacements of alpha carbon atoms were found around the mutation points. In the model of the double mutant (A88V/A98T), on the other hand, the structural changes around the mutation point counterbalanced each other, and thus no crucial displacements occurred. This mutual effect may be related to the enhanced activity of the double mutant.     

Improving the activity and stability of GL-7-ACA acylase CA130 by site-directed mutagenesis

In the present study, glutaryl-7-amino cephalosporanic acid acylase from Pseudomonas sp. strain 130 (CA130) was mutated to improve its enzymatic activity and stability. Based on the crystal structure of CA130, two series of amino acid residues, one from those directly involved in catalytic function and another from those putatively involved in surface charge, were selected as targets for site-directed mutagenesis. In the first series of experiments, several key residues in the substrate-binding pocket were substituted, and the genes were expressed in Escherichia coli for activity screening. Two of the mutants constructed, Y151alphaF and Q50betaN, showed two- to threefold-increased catalytic efficiency (k(cat)/K(m)) compared to wild-type CA130. Their K(m) values were decreased by ca. 50%, and the k(cat) values increased to 14.4 and 16.9 s(-1), respectively. The ability of these mutants to hydrolyze adipoyl 6-amino penicillinic acid was also improved. In the second series of mutagenesis, several mutants with enhanced stabilities were identified. Among them, R121betaA and K198betaA had a 30 to 58% longer half-life than wild-type CA130, and K198betaA and D286betaA showed an alkaline shift of optimal pH by about 1.0 to 2.0 pH units. To construct an engineered enzyme with the properties of both increased activity and stability, the double mutant Q50betaN/K198betaA was expressed. This enzyme was purified and immobilized for catalytic analysis. The immobilized mutant enzyme showed a 34.2% increase in specific activity compared to the immobilized wild-type CA130.     

枯草杆菌蛋白酶E的156和165位突变

应用定点突变方法,在M222A突变的枯草杆菌蛋白酶E基因上进行E156S和V165I定点突变. 将突变基因插入大肠杆菌-枯草杆菌穿梭质粒pBE-2中,在碱性和中性蛋白酶缺陷型的枯草杆菌DB104中进行表达,得到突变种(M222A,E156S)和(M222A,E156S,V165I)蛋白酶E. 性质测定表明,E156S突变使蛋白酶比活力增加90%,并不影响酶的热稳定性和抗氧化性. 而V165I突变使蛋白酶比活力降低.     

Structural and catalytic similarities between nucleotide pyrophosphatases/phosphodiesterases and alkaline phosphatases

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) generate nucleoside 5'-monophosphates from a variety of nucleotides and their derivatives. Here we show by data base analysis that these enzymes are conserved from eubacteria to higher eukaryotes. We also provide evidence for the existence of two additional members of the mammalian family of ecto-NPPs. Homology searches and alignment-assisted mutagenesis revealed that the catalytic core of NPPs assumes a fold similar to that of a superfamily of phospho-/sulfo-coordinating metalloenzymes comprising alkaline phosphatases, phosphoglycerate mutases, and arysulfatases. Mutation of mouse NPP1 in some of its predicted metal-coordinating residues (D358N or H362Q) or in the catalytic site threonine (T238S) resulted in an enzyme that could still form the nucleotidylated catalytic intermediate but was hampered in the second step of catalysis. We also obtained data indicating that the ability of some mammalian NPPs to auto(de)phosphorylate is due to an intrinsic phosphatase activity, whereby the enzyme phosphorylated on Thr-238 represents the covalent intermediate of the phosphatase reaction. The results of site-directed mutagenesis suggested that the nucleotide pyrophosphatase/phosphodiesterase and the phosphatase activities of NPPs are mediated by a single catalytic site.     

Identification of catalytically important residues in the active site of Escherichia coli transaldolase.

The roles of invariant residues at the active site of transaldolase B from Escherichia coli have been probed by site-directed mutagenesis. The mutant enzymes D17A, N35A, E96A, T156A, and S176A were purified from a talB-deficient host and analyzed with respect to their 3D structure and kinetic behavior. X-ray analysis showed that side chain replacement did not induce unanticipated structural changes in the mutant enzymes. Three mutations, N35A, E96A, and T156A resulted mainly in an effect on apparent kcat, with little changes in apparent Km values for the substrates. Residues N35 and T156 are involved in the positioning of a catalytic water molecule at the active site and the side chain of E96 participates in concert with this water molecule in proton transfer during catalysis. Substitution of Ser176 by alanine resulted in a mutant enzyme with 2.5% residual activity. The apparent Km value for the donor substrate, fructose 6-phosphate, was increased nearly fivefold while the apparent Km value for the acceptor substrate, erythrose 4-phosphate remained unchanged, consistent with a function for S176 in the binding of the C1 hydroxyl group of the donor substrate. The mutant D17A showed a 300-fold decrease in kcat, and a fivefold increase in the apparent Km value for the acceptor substrate erythrose 4-phosphate, suggesting a role of this residue in carbon-carbon bond cleavage and stabilization of the carbanion/enamine intermediate.     

Expression and purification of his-tagged rat peroxisomal acyl-CoA oxidase I wild-type and E421 mutant proteins

Rat peroxisomal acyl-CoA oxidase I is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patients has been previously reported. We cloned the gene of rat peroxisomal acyl-CoA oxidase I into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5' end of the gene. The cloned gene was overexpressed in Escherichia coli and the soluble protein was purified with a nickel HiTrap chelating metal-affinity column in 90% yield to apparent homogeneity. The specific activity of the purified His-tagged rat peroxisomal acyl-CoA oxidase I was 1.5 micromol/min/mg. It has been proposed that Glu421 is a catalytic residue responsible for deprotonation of alpha-proton of acyl-CoA substrate. We constructed four mutant expression plasmids of the enzyme, pACO(E421D), pACO(E421A), pACO(E421Q), and pACO(E421G) using site-directed mutagenesis. Mutant proteins were overexpressed in E. coli and purified with a nickel metal-affinity column. Kinetic studies of wild-type and mutant proteins were carried out, and the result confirmed that Glu421 is a catalytic residue of rat peroxisomal acyl-CoA oxidase I. Our overexpression in E. coli and one-step purification of the highly active N-terminal His-tagged rat peroxisomal acyl-CoA oxidase I greatly facilitated our further investigation of this enzyme, and our result from site-directed mutagenesis increased our understanding of the mechanism for the reaction catalyzed by peroxisomal acyl-CoA oxidase I.     

The crystal structure of three site-directed mutants of Escherichia coli dihydrodipicolinate synthase: further evidence for a catalytic triad

Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) catalyses the branchpoint reaction of lysine biosynthesis in plants and microbes: the condensation of (S)-aspartate-beta-semialdehyde and pyruvate. The crystal structure of wild-type DHDPS has been published to 2.5A, revealing a tetrameric molecule comprised of four identical (beta/alpha)(8)-barrels, each containing one active site. Previous workers have hypothesised that the catalytic mechanism of the enzyme involves a catalytic triad of amino acid residues, Tyr133, Thr44 and Tyr107, which provide a proton shuttle to transport protons from the active site to solvent. We have tested this hypothesis using site-directed mutagenesis to produce three mutant enzymes: DHDPS-Y133F, DHDPS-T44V and DHDPS-Y107F. Each of these mutants has substantially reduced activity, consistent with the catalytic triad hypothesis. We have determined each mutant crystal structure to at least 2.35A resolution and compared the structures to the wild-type enzyme. All mutant enzymes crystallised in the same space group as the wild-type form and only minor differences in structure are observed. These results suggest that the catalytic triad is indeed in operation in wild-type DHDPS.     

Importance of the conserved residues in the peptidoglycan glycosyltransferase module of the class A penicillin-binding protein 1b of Escherichia coli

The peptidoglycan glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs catalyze glycan chain elongation of the bacterial cell wall. These enzymes belong to the GT51 family, are characterized by five conserved motifs, and have some fold similarity with the phage lambda lysozyme. In this work, we have systematically modified all the conserved amino acid residues of the GT module of Escherichia coli class A PBP1b by site-directed mutagenesis and determined their importance for the in vivo and in vitro activity and the thermostability of the protein. To get an insight into the GT active site of this paradigm enzyme, a model of PBP1b GT domain was constructed based on the available crystal structures (PDB codes 2OLV and 2OLU). The data show that in addition to the essential glutamate residues Glu233 of motif 1 and Glu290 of motif 3, the residues Phe237 and His240 of motif 1 and Gly264, Thr267, Gln271, and Lys274 of motif 2, all located in the catalytic cavity of the GT domain, are essential for the in vitro enzymatic activity of the PBP1b and for its in vivo functioning. Thus, the first three conserved motifs contain most of the residues that are required for the GT activity of the PBP1b. The residues Asp234, Phe237, His240, Thr267, and Gln271 are proposed to maintain the structure of the active site and the positioning of the catalytic Glu233.     

Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis

Despite the structural similarities between cholesterol oxidase from Streptomyces and that from Brevibacterium, both enzymes exhibit different characteristics, such as catalytic activity, optimum pH and temperature. In attempts to define the molecular basis of differences in catalytic activity or stability, substitutions at six amino acid residues were introduced into cholesterol oxidase using site-directed mutagenesis of its gene. The amino acid substitutions chosen were based on structural comparisons of cholesterol oxidases from Streptomyces and BREVIBACTERIUM: Seven mutant enzymes were constructed with the following amino acid substitutions: L117P, L119A, L119F, V145Q, Q286R, P357N and S379T. All the mutant enzymes exhibited activity with the exception of that with the L117P mutation. The resulting V145Q mutant enzyme has low activities for all substrates examined and the S379T mutant enzyme showed markedly altered substrate specificity compared with the wild-type enzyme. To evaluate the role of V145 and S379 residues in the reaction, mutants with two additional substitutions in V145 and four in S379 were constructed. The mutant enzymes created by the replacement of V145 by Asp and Glu had much lower catalytic efficiency for cholesterol and pregnenolone as substrates than the wild-type enzyme. From previous studies and this study, the V145 residue seems to be important for the stability and substrate binding of the cholesterol oxidase. In contrast, the catalytic efficiencies (k(cat)/K(m)) of the S379T mutant enzyme for cholesterol and pregnenolone were 1.8- and 6.0-fold higher, respectively, than those of the wild-type enzyme. The enhanced catalytic efficiency of the S379T mutant enzyme for pregnenolone was due to a slightly high k(cat) value and a low K(m) value. These findings will provide several ideas for the design of more powerful enzymes that can be applied to clinical determination of serum cholesterol levels and as sterol probes.     

Role of cysteine residues in Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA reductase. Site-directed mutagenesis and characterization of the mutant enzymes

Each of the four identical subunits of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase contains two cysteine residues, Cys156 and Cys296 (Beach, M. J., and Rodwell, V. W. (1989) J. Bacteriol. 171, 2994-3001). Both are accessible to modification by sulfhydryl reagents under nondenaturing conditions (Jordan-Starck, T. C., and Rodwell, V. W. (1989) J. Biol. Chem. 264, 17913-17918). We used site-directed mutagenesis to construct three mutant enzymes in which alanine replaced either or both cysteine residues. Mutant enzymes C156A, C296A, and C156/296A were over-expressed in Escherichia coli and were found to be fully active. Following their purification, all four forms of the enzyme were compared with respect to their catalytic efficiency, their affinities for the substrates of all four catalyzed reactions, and for their sensitivity to inactivation by sulfhydryl reagents. Replacement of cysteine residues with alanine residues had no major effect on either the specific activity or the affinity of the enzymes for any substrate. The mutants catalyzed all four HMG-CoA reductase reactions as efficiently as did the wild-type enzyme, and coenzyme A stimulated mevaldehyde reduction to the same extent as for wild-type HMG-CoA reductase. Mutant C156A and the cysteine-free mutant C156/296A were not inactivated by 5,5'-dithiobis(2-nitrobenzoate). By contrast, mutant C296A was inactivated to the same extent as was the wild-type enzyme. Following treatment of the mutant enzymes with N-ethylmaleimide, the four reductase reactions catalyzed by mutant C296A were inactivated to the same extent as for the wild-type enzyme. Neither mutant C156A nor C156/296A was affected by this reagent. We conclude that the sulfhydryl reagent-reactive group whose derivatization leads to loss of enzymatic activity is Cys156. However, this residue is not an essential active site residue since neither substrate binding nor catalysis was affected when it was replaced by alanine. Possible roles of cysteine in maintaining structural stability are discussed.     

Role of the Conserved Thr399 and Thr417 Residues of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> γ-Glutamyltranspeptidase as Evaluated by Mutational Analysis

Role of the conserved Thr399 and Thr417 residues of Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) was investigated by site-directed mutagenesis. Substitutions of Thr399 and Thr417 of BlGGT with Ser resulted in a dramatic reduction in enzymatic activity. A complete loss of the GGT activity was observed in T399A, T399C, T417A, and T417K mutant enzymes. Furthermore, mutations on these two residues impaired the capability of autocatalytic processing of the enzyme. In vitro maturation experiments showed that BlGGT mutant precursors, pro-T399S, pro-T417S, and pro-T417A, could precede a time-dependent autocatalytic process to generate the 44.9- and 21.7-kDa subunits; however, the processed T417A had no enzymatic activity. Measurement of intrinsic tryptophan fluorescence revealed alteration of the microenvironment of aromatic amino acid residues, while Far-UV circular dichroism spectra were nearly identical for wild-type and mutant enzymes. These results suggest that residues Thr399 and Thr417 are important for BlGGT in the enzymatic maturation and reaction. An erratum to this article can be found at     

Functional analysis of mutations in the PIS gene, which encodes Saccharomyces cerevisiae phosphatidylinositol synthase

Abstract The PIS gene for an enzyme phosphatidylinositol synthase having an increased K _m for myo-inositol, was isolated from Saccharomyces cerevisiae . The mutant PIS gene contained a CAA codon at position 114 instead of the CAC codon observed in the wild-type gene, resulting in alteration of the amino acid from His to Gln. Oligonucleotide mediated site-directed mutagenesis of PIS at codon 114 revealed that mutant genes with codons for Ala, Thr and Leu could support yeast cell growth in vivo, but those for Asp, Lys and Tyr could not. All mutant enzymes when expressed in Escherichia coli showed greatly reduced in vitro activity.     

Characterization of a major secretory protein in the cane toad (Bufo marinus) choroid plexus as an amphibian lipocalin-type prostaglandin D synthase

Here we report the enzymatic and ligand-binding properties of a major secretory protein in the choroid plexus of cane toad, Bufo marinus, whose protein is homologous with lipocalin-type prostaglandin (PG) D synthase (L-PGDS) and is recombinantly expressed in Xenopus A6 cells and Escherichia coli. The toad protein bound all-trans retinal, bile pigment, and thyroid hormones with high affinities (K(d)=0.17 to 2.00 microM). The toad protein also catalysed the L-PGDS activity, which was accelerated in the presence of GSH or DTT, similar to the mammalian enzyme. The K(m) value for PGH(2) (17 microM) of the toad protein was almost the same as that of rat L-PGDS (14 microM), whereas the turnover number (6 min(-1)) was approximately 28 fold lower than that of rat L-PGDS. Site-directed mutagenesis based on a modeled structure of the toad protein revealed that Cys(59) and Thr(61) residues were crucial for the PGDS activity. The quadruple Gly(39)Ser/Ala(75)Ser/Ser(140)Thr/Phe(142)Tyr mutant of the toad protein, resembling mouse L-PGDS, showed a 1.6 fold increase in the turnover number and a shift in the optimum pH for the PGDS activity from 9.0 to 8.5. Our results suggest that the toad protein is a prototype of L-PGDS with a highly functional ligand-binding pocket and yet with a primitive catalytic pocket.     

Replacement of arginine-171 and aspartate-453 in Streptomyces coelicolor malate synthase A by site-directed mutagenesis inactivates the enzyme

Malate synthase, a key enzyme of the glyoxylate cycle, catalyzes the condensation of glyoxylate and acetyl-CoA to yield malate and CoA. Escherichia coli is known to possess two forms of malate synthase, A and G respectively. The recent elucidation of the E. coli malate synthase G crystal structure suggested two residues, Arg338 and Asp631, are essential for catalysis. Multiple sequence alignment of 26 known malate synthase enzymes revealed that the two proposed sites are highly conserved, despite the low homologies between the two distinct forms of the enzyme (13-18%). The conservation of these residues in both forms of malate synthase suggests that they possess a similar catalytic strategy. Thus, despite the absence of a three-dimensional structure for malate synthase A, the significance of this enzyme in the primary metabolic pathway has prompted the investigation of the involvement of the corresponding residues, Arg171 and Asp453, in Streptomyces coelicolor malate synthase A by site-directed mutagenesis. Heterologous expression in E. coli followed by purification of the constructed mutant proteins, Arg171Leu and Asp453Ala, were performed and subsequent enzyme assays of the purified mutant proteins indicated a significant loss of catalytic activity, thus attesting to the need for the corresponding conserved residues to maintain malate synthase functionality.