Aspzincin, a family of metalloendopeptidases with a new zinc-binding motif. Identification of new zinc-binding sites (His(128), His(132), and Asp(164)) and three catalytically crucial residues (Glu(129), Asp(143), and Tyr(106)) of deuterolysin from Aspergillus oryzae by site-directed mutagenesis.

Deuterolysin (EC 3.4.24.39; formerly designated as neutral proteinase II) from Aspergillus oryzae, which contains 1 g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. Active-site determination of the recombinant enzyme expressed in Escherichia coli was performed by site-directed mutagenesis. Substitutions of His(128) and His(132) with Arg, of Glu(129) with Gln or Asp, of Asp(143) with Asn or Glu, of Asp(164) with Asn, and of Tyr(106) with Phe resulted in almost complete loss of the activity of the mutant enzymes. It can be concluded that His(128), His(132), and Asp(164) provide the Zn(2+) ligands of the enzyme according to a (65)Zn binding assay. Based on site-directed mutagenesis experiments, it was demonstrated that the three essential amino acid residues Glu(129), Asp(143), and Tyr(106) are catalytically crucial residues in the enzyme. Glu(129) may be implicated in a central role in the catalytic function. We conclude that deuterolysin is a member of a family of Zn(2+) metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH + D" motif and an aspartic acid as the third zinc ligand.     

Mechanism of action of Escherichia coli ribonuclease III. Stringent chemical requirement for the glutamic acid 117 side chain and Mn2+ rescue of the Glu117Asp mutant

Escherichia coli ribonuclease III (EC 3.1.24) is a double-strand- (ds-) specific endoribonuclease involved in the maturation and decay of cellular, phage, and plasmid RNAs. RNase III is a homodimer and requires Mg(2+) to hydrolyze phosphodiesters. The RNase III polypeptide contains an N-terminal catalytic (nuclease) domain which exhibits eight highly conserved acidic residues, at least one of which (Glu117) is important for phosphodiester hydrolysis but not for substrate binding [Li and Nicholson (1996) EMBO J. 15, 1421-1433]. To determine the side chain requirements for activity, Glu117 was changed to glutamine or aspartic acid. The mutant proteins were purified as (His)(6)-tagged species, and both exhibited normal homodimeric behavior as shown by chemical cross-linking. The Glu117Gln mutant is unable to cleave substrate in vitro under all tested conditions but can bind substrate. The Glu117Asp mutant also is defective in cleavage while able to bind substrate. However, low level activity is observed at extended reaction times and high enzyme concentrations, with an estimated catalytic efficiency approximately 15 000-fold lower than that of RNase III. The activity of the Glu117Asp mutant but not that of the Glu117Gln mutant can be greatly enhanced by substituting Mn(2+) for Mg(2+), with the catalytic efficiency of the Glu117Asp-Mn(2+) holoenzyme approximately 400-fold lower than that of the RNase III-Mn(2+) holoenzyme. For RNase III, a Mn(2+) concentration of 1 mM provides optimal activity, while concentrations >5 mM are inhibitory. In contrast, the Glu117Asp mutant is not inhibited by high concentrations of Mn(2+). Finally, high concentrations of Mg(2+) do not inhibit RNase III nor relieve the Mn(2+)-dependent inhibition. In summary, these experiments establish the stringent functional requirement for a precisely positioned carboxylic acid group at position 117 and reveal two classes of divalent metal ion binding sites on RNase III. One site binds either Mg(2+) or Mn(2+) and supports catalysis, while the other site is specific for Mn(2+) and confers inhibition. Glu117 is important for the function of both sites. The implications of these findings on the RNase III catalytic mechanism are discussed.     

Functional consequences of single amino acid substitutions in calmodulin-activated adenylate cyclase of Bordetella pertussis.

Calmodulin-activated adenylate cyclase of Bordetella pertussis and Bacillus anthracis are two cognate bacterial toxins. Three short regions of 13-24 amino acid residues in these proteins exhibit between 66 and 80% identity. Site-directed mutagenesis of four residues in B. pertussis adenylate cyclase situated in the second (Asp188, Asp190) and third (His298, Glu301) segments of identity were accompanied by important decrease, or total loss, of enzyme activity. The calmodulin-binding properties of mutated proteins showed no important differences when compared to the wild-type enzyme. Apart from the loss of enzymatic activity, the most important change accompanying replacement of Asp188 by other amino acids was a dramatic decrease in binding of 3'-anthraniloyl-2'-deoxyadenosine 5'-triphosphate, a fluorescent analogue of ATP. From these results we concluded that the two neighbouring aspartic acid residues in B. pertussis adenylate cyclase, conserved in many other ATP-utilizing enzymes, are essential for binding the Mg(2+)-nucleotide complex, and for subsequent catalysis. Replacement of His298 and Glu301 by other amino acid residues affected the nucleotide-binding properties of adenylate cyclase to a lesser degree suggesting that they might be important in the mechanism of enzyme activation by calmodulin, rather than being involved directly in catalysis.     

The HELLGH motif of rat liver dipeptidyl peptidase III is involved in zinc coordination and the catalytic activity of the enzyme.

The role of the HELLGH (residues 450-455) motif in the sequence of rat dipeptidyl peptidase III (EC 3.4.14.4) was investigated by replacing Glu451 with an alanine or an aspartic acid residue and by replacing His450 and His455 with a tyrosine residue by site-directed mutagenesis. Mutated cDNAs were expressed three or four times in Escherichia coli, and the resulting proteins were purified to apparent homogeneity. None of the expressed mutated proteins exhibited DPP III activity. The mutants of Glu451 contained 1 mol of zinc per mole of protein, but mutants His450 and His455 did not contain significant amounts of zinc as determined by atomic absorption spectrometry. The Leu453-deleted enzyme (having the zinc aminopeptidase motif HExxH-18-E) had almost the same order of binding affinity (for Arg-Arg-2-naphthylamide) as the wild-type enzyme, but the specificity constant was about 10%. These results provide evidence that the suitable number of amino acids included between Glu451 and His455 is three residues for the enzyme activity and confirm that residues His450, His455, and Glu451 are involved in zinc coordination and catalytic activity.     

Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase. Mutagenesis at metal site 1

Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate (OAA) and ATP from PEP, ADP and CO(2). Mutations of PEP carboxykinase have been constructed where the residues His(225) and Asp(263), two residues of the enzyme's putative Mn(2+) binding site, were altered. Kinetic studies of the His225Glu, and Asp263Glu PEP carboxykinases show 600- and 16,800-fold reductions in V(max) relative to the wild-type enzyme, respectively, with minor alterations in K(m) for Mn(2+). Molecular modeling of wild-type and mutant enzymes suggests that the lower catalytic efficiency of the Asp263Glu enzyme could be explained by a movement of the lateral chain of Lys(248), a critical catalytic residue, away from the reaction center. The effect on catalysis of introducing a negatively charged oxygen atom in place of N(epsilon-2) at position 225 is discussed in terms of altered binding energy of the intermediate enolpyruvate.     

Novel bifunctional hyperthermostable carboxypeptidase/aminoacylase from Pyrococcus horikoshii OT3

Genome sequencing of the thermophilic archaeon Pyrococcus horikoshii OT3 revealed a gene which had high sequence similarity to the gene encoding the carboxypeptidase of Sulfolobus solfataricus and also to that encoding the aminoacylase from Bacillus stearothermophilus. The gene from P. horikoshii comprises an open reading frame of 1,164 bp with an ATG initiation codon and a TGA termination codon, encoding a 43,058-Da protein of 387 amino acid residues. However, some of the proposed active-site residues for carboxypeptidase were not found in this gene. The gene was overexpressed in Escherichia coli with the pET vector system, and the expressed enzyme had high hydrolytic activity for both carboxypeptidase and aminoacylase at high temperatures. The enzyme was stable at 90 degrees C, with the highest activity above 95 degrees C. The enzyme contained one bound zinc ion per one molecule that was essential for the activity. The results of site-directed mutagenesis of Glu367, which corresponds to the essential Glu270 in bovine carboxypeptidase A and the essential Glu in other known carboxypeptidases, revealed that Glu367 was not essential for this enzyme. The results of chemical modification of the SH group and site-directed mutagenesis of Cys102 indicated that Cys102 was located at the active site and was related to the activity. From these findings, it was proven that this enzyme is a hyperthermostable, bifunctional, new zinc-dependent metalloenzyme which is structurally similar to carboxypeptidase but whose hydrolytic mechanism is similar to that of aminoacylase. Some characteristics of this enzyme suggested that carboxypeptidase and aminoacylase might have evolved from a common origin.     

Lipoamide dehydrogenase from Azotobacter vinelandii: site-directed mutagenesis of the His450-Glu455 diad. Kinetics of wild-type and mutated enzymes.

Three amino acid residues in the active site of lipoamide dehydrogenase from Azotobacter vinelandii were replaced with other residues. His450, the active-site base, was replaced with Ser, Tyr or Phe. Pro451, from X-ray analysis found to be in cis conformation positioning the backbone carbonyl of His450 close to N3 of the flavin, was changed to Ala. Glu455, from X-ray analysis expected to be involved in modulating the pKa of the base (His450), was replaced with Asp and Gln. The general conclusion is that mutation of the His-Glu diad impairs intramolecular electron transfer between the disulfide/dithiol and the FADH-/FAD. The wild-type enzyme functions according to a ping-pong mechanism in the physiological reaction in which the formation of NADH is rate-limiting. Above pH 8.0 the enzyme is strongly inhibited by the product NADH. The pH dependence of the steady-state kinetics using the NAD+ analog 3-acetylpyridine adenine dinucleotide (AcPyAde+) reveals a pKa of 8.1 in the pKm AcPyAde+ plot indicating that this pKa is related to the deprotonation of His450 [Benen, J., Berkel van, W., Zak, Z., Visser, T., Veeger, C. & Kok de, A. (1991) Eur. J. Biochem. 202, 863-872] and to the inhibition by NADH. The mutations considerably affect turnover. Enzymes with the mutations Pro451----Ala, His450----Phe and His450----Tyr appear to be almost inactive in both directions. Enzyme His450----Ser is minimally active, V at the pH optimum being 0.5% of wild-type activity in the physiological reaction. Rapid reaction kinetics show that for the His450-mutated enzymes the reductive half reaction using reduced 6,8-thioctic acid amide [Lip(SH)2] is rate-limiting and extremely slow when compared using reduced 6,8-thioctic acid amide [Lip(SH)2] is rate-limiting and extremely slow when compared to the wild-type enzyme. For enzyme Pro451----Ala it is concluded that the loss of activity is due to over-reduction by Lip(SH)2 and NADH. The Glu455-mutated enzymes are catalytically competent but show strong inhibition by the product NADH (enzyme Glu455----Asp more than Glu455----Gln). The inhibition can largely be overcome by using AcPyAde+ instead of NAD+ in the physiological reaction. The rapid reaction kinetics obtained for enzymes Glu455----Asp and Glu455----Gln deviate from the wild-type enzyme. It is concluded that this difference is due to cooperativity between the active sites in this dimeric enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Catalytic mechanism of endonuclease v: a catalytic and regulatory two-metal model

The enzyme endonuclease V initiates repair of deaminated DNA bases by making an endonucleolytic incision on the 3' side one nucleotide from a base lesion. In this study, we have used site-directed mutagenesis to characterize the role of the highly conserved residues D43, E89, D110, and H214 in Thermotoga maritima endonuclease V catalysis. DNA cleavage and Mn(2+)-rescue analysis suggest that amino acid substitutions at D43 impede the enzymatic activity severely while mutations at E89 and D110 may be tolerated. Mutations at H214 yield enzyme that maintains significant DNA cleavage activity. The H214D mutant exhibits little change in substrate specificity or DNA cleavage kinetics, suggesting the exchangeability between His and Asp at this site. DNA binding analysis implicates the involvement of the four residues in metal binding. Mn(2+)-mediated cleavage of inosine-containing DNA is stimulated by the addition of Ca(2+), a metal ion that does not support catalysis. The effects of Mn(2+) on Mg(2+)-mediated DNA cleavage show a complexed initial stimulatory and later inhibitory pattern. The data obtained from the dual metal ion analyses lead to the notion that two metal ions are involved in endonuclease V-mediated catalysis. A catalytic and regulatory two-metal model is proposed.     

Isomaltulose synthase from Klebsiella sp. strain LX3: gene cloning and characterization and engineering of thermostability

The gene (palI) encoding isomaltulose synthase (PalI) from a soil bacterial isolate, Klebsiella sp. strain LX3, was cloned and characterized. PalI converts sucrose into isomaltulose, trehalulose, and trace amounts of glucose and fructose. Sequence domain analysis showed that PalI contains an alpha-amylase domain and (beta/alpha)(8)-barrel structures, suggesting that it belongs to the alpha-amylase family. Sequence alignment indicated that the five amino acid residues of catalytic importance in alpha-amylases and glucosyltransferases (Asp(241), Glu(295), Asp(369), His(145), and His(368)) are conserved in PalI. Purified recombinant PalI displayed high catalytic efficiency, with a Km of 54.6 +/- 1.7 mM for sucrose, and maximum activity (approximately 328.0 +/- 2.5 U/mg) at pH 6.0 and 35 degrees C. PalI activity was strongly inhibited by Fe3+ and Hg2+ and was enhanced by Mn2+ and Mg2+. The half-life of PalI was 1.8 min at 50 degrees C. Replacement of selected amino acid residues by proline significantly increased the thermostability of PalI. Simultaneous replacement of Glu(498) and Arg(310) with proline resulted in an 11-fold increase in the half-life of PalI at 50 degrees C.     

A mutagenic study of beta-1,4-galactosyltransferases from Neisseria meningitidis

N-terminal His-tagged recombinant beta-1,4-galactosyltransferase from Neisseria meningitidis was expressed and purified to homogeneity by column chromatography using Ni-NTA resin. Mutations were introduced to investigate the roles of, Ser68, His69, Glu88, Asp90, and Tyr156, which are components of a highly conserved region in recombinant beta-1,4 galactosyltransferase. Also, the functions of three other cysteine residues, Cys65, Cys139, and Cys205, were investigated using site-directed mutagenesis to determine the location of the disulfide bond and the role of the sulfhydryl groups. Purified mutant galactosyltransferases, His69Phe, Glu88Gln and Asp90Asn completely shut down wild-type galactosyltransferase activity (1-3 %). Also, Ser68Ala showed much lower activity than wild-type galactosyltransferase (19 %). However, only the substitution of Tyr156Phe resulted in a slight reduction in galactosyltransferase activity (90 %). The enzyme was found to remain active when the cysteine residues at positions 139 and 205 were replaced separately with serine. However, enzyme reactivity was found to be markedly reduced when Cys65 was replaced with serine (27 %). These results indicate that conserved amino acids such as Cys65, Ser68, His69, Glu88, and Asp90 may be involved in the binding of substrates or in the catalysis of the galactosyltransferase reaction.     

Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

Genome sequencing of the thermophilic archaeon Pyrococcus horikoshii OT3 revealed a gene which had high sequence similarity to the gene encoding the carboxypeptidase of Sulfolobus solfataricus and also to that encoding the aminoacylase from Bacillus stearothermophilus. The gene from P. horikoshii comprises an open reading frame of 1,164 bp with an ATG initiation codon and a TGA termination codon, encoding a 43,058-Da protein of 387 amino acid residues. However, some of the proposed active-site residues for carboxypeptidase were not found in this gene. The gene was overexpressed in Escherichia coli with the pET vector system, and the expressed enzyme had high hydrolytic activity for both carboxypeptidase and aminoacylase at high temperatures. The enzyme was stable at 90°C, with the highest activity above 95°C. The enzyme contained one bound zinc ion per one molecule that was essential for the activity. The results of site-directed mutagenesis of Glu367, which corresponds to the essential Glu270 in bovine carboxypeptidase A and the essential Glu in other known carboxypeptidases, revealed that Glu367 was not essential for this enzyme. The results of chemical modification of the SH group and site-directed mutagenesis of Cys102 indicated that Cys102 was located at the active site and was related to the activity. From these findings, it was proven that this enzyme is a hyperthermostable, bifunctional, new zinc-dependent metalloenzyme which is structurally similar to carboxypeptidase but whose hydrolytic mechanism is similar to that of aminoacylase. Some characteristics of this enzyme suggested that carboxypeptidase and aminoacylase might have evolved from a common origin.     

Physico-chemical properties of the epoxide hydrolase from Rhodosporidium toruloides

Residue-specific chemical modification of amino acid residues of the microsomal epoxide hydrolase (mEH) from Rhodosporidium toruloides UOFS Y-0471 revealed that the enzyme is inactivated through modification of Asp/Glu and His residues, as well as through modification of Ser. Since Asp acts as the nucleophile, and Asp/Glu and His serve as charge relay partners in the catalytic triad of microsomal and soluble epoxide hydrolases during epoxide hydrolysis, inactivation of the enzyme by modification of the Asp/Glu and His residues agrees with the established reaction mechanism of these enzymes. However, the inactivation of the enzyme through modification of Ser residues is unexpected, suggesting that a Ser in the catalytic site is indispensable for substrate binding by analogy of the role of Ser residues in the related L-2-haloacid dehalogenases, as well as the ATPase and phosphatase enzymes. Co²⁺, Hg²⁺, Ag⁺, Mg²⁺ and Ca²⁺ inhibited enzyme activity and EDTA increased enzyme activity. The activation energy for inactivation of the enzyme was 167 kJ mol^–1. Kinetic constants for the enzyme could not be determined since unusual behaviour was displayed during hydrolysis of 1,2-epoxyoctane by the purified enzyme. Enantioselectivity w as strongly dependent on substrate concentration. When the substrate was added in concentrations ensuring two-phase conditions, the enantioselectivity was greatly enhanced. On the basis of these results, it is proposed that this enzyme acts at an interface, analogous to lipases.     

Expression and characterization of the biofilm-related and carnosine-hydrolyzing aminoacylhistidine dipeptidase from Vibrio alginolyticus

The biofilm-related and carnosine-hydrolyzing aminoacylhistidine dipeptidase (pepD) gene from Vibrio alginolyticus was cloned and sequenced. The recombinant PepD protein was produced and biochemically characterized and the putative active-site residues responsible for metal binding and catalysis were identified. The recombinant enzyme, which was identified as a homodimeric dipeptidase in solution, exhibited broad substrate specificity for Xaa-His and His-Xaa dipeptides, with the highest activity for the His-His dipeptide. Sequence and structural homologies suggest that the enzyme is a member of the metal-dependent metallopeptidase family. Indeed, the purified enzyme contains two zinc ions per monomer. Reconstitution of His.Tag-cleaved native apo-PepD with various metal ions indicated that enzymatic activity could be optimally restored when Zn2+ was replaced with other divalent metal ions, including Mn2+, Co2+, Ni2+, Cu2+ and Cd2+, and partially restored when Zn2+ was replaced with Mg2+. Structural homology modeling of PepD also revealed a 'catalytic domain' and a 'lid domain' similar to those of the Lactobacillus delbrueckii PepV protein. Mutational analysis of the putative active-site residues supported the involvement of His80, Asp119, Glu150, Asp173 and His461 in metal binding and Asp82 and Glu149 in catalysis. In addition, individual substitution of Glu149 and Glu150 with aspartic acid resulted in the partial retention of enzymatic activity, indicating a functional role for these residues on the catalysis and zinc ions, respectively. These effects may be necessary either for the activation of the catalytic water molecule or for the stabilization of the substrate-enzyme tetrahedral intermediate. Taken together, these results may facilitate the design of PepD inhibitors for application in antimicrobial treatment and antibody-directed enzyme prodrug therapy.     

The molecular basis of aminoacylase 1 deficiency

Aminoacylase 1 is a zinc-binding enzyme which hydrolyzes N-acetyl amino acids into the free amino acid and acetic acid. Deficiency of aminoacylase 1 due to mutations in the aminoacylase 1 (ACY1) gene follows an autosomal-recessive trait of inheritance and is characterized by accumulation of N-acetyl amino acids in the urine. In affected individuals neurological findings such as febrile seizures, delay of psychomotor development and moderate mental retardation have been reported. Except for one missense mutation which has been studied in Escherichia coli, mutations underlying aminoacylase 1 deficiency have not been characterized so far. This has prompted us to approach expression studies of all mutations known to occur in aminoacylase 1 deficient individuals in a human cell line (HEK293), thus providing the authentic human machinery for posttranslational modifications. Mutations were inserted using site directed mutagenesis and aminoacylase 1 enzyme activity was assessed in cells overexpressing aminoacylase 1, using mainly the natural high affinity substrate N-acetyl methionine. Overexpression of the wild type enzyme in HEK293 cells resulted in an approximately 50-fold increase of the aminoacylase 1 activity of homogenized cells. Most mutations resulted in a nearly complete loss of enzyme function. Notably, the two newly discovered mutations p.Arg378Trp, p.Arg378Gln and the mutation p.Arg393His yielded considerable residual activity of the enzyme, which is tentatively explained by their intramolecular localization and molecular characteristics. In contrast to aminoacylase 1 variants which showed no detectable aminoacylase 1 activity, aminoacylase 1 proteins with the mutations p.Arg378Trp, p.Arg378Gln and p.Arg393His were also detected in Western blot analysis. Investigations of the molecular bases of additional cases of aminoacylase 1 deficiency contribute to a better understanding of this inborn error of metabolism whose clinical significance and long-term consequences remain to be elucidated.     

Chemical investigations on pig kidney aminoacylase.

1. Preparations of purified pig kidney aminoacylase (N-Acylamino-acid amidohydrolase, EC 3.5.1.14) were obtained by Sephadex and DEAE-cellulose chromatography in homogeneous form as judged by polyacrylamide gel electrophoresis and immunoelectrophoresis. 2. The apparent molecular weight of the enzyme, determined by gel filtration, was about 86 000. After treatment with mercaptoethanol, performic acid or sodium dodecyl sulphate a band with an apparent molecular weight of approximately 43 000 was observed in polyacrylamide gels containing sodium dodecyl sulphate. Thus pig kidney aminoacylase seems to be composed of two subunits. 3. The amino acid composition of the enzyme was determined. Aminoacylase contains 772 amino acids, which corresponds to a molecular weight of 85 500. 12 tryptophan and 12 half-cystine residues were found. 4. Each subunit of the enzyme contains two -SH groups of different reactivity and two disulfide bonds one of which is easily cleaved by -SH compounds, the second only by performic acid oxidation. 5. Chemical modification of two -SH groups abolishes the catalytic activity of aminoacylase. Cleavage of two disulfide bonds also inactivates the enzyme. It is suggested that the enzyme has two active sites each containing an essential -SH group and disulfide bond. One active site is assumed to be part of each subunit.     

Site-directed mutagenesis of the bacterial metalloamidase UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC). Identification of the zinc binding site

UDP-3-O-(acyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the second step in the biosynthesis of lipid A in Gram-negative bacteria. Compounds targeting this enzyme are proposed to chelate the single, essential zinc ion bound to LpxC and have been demonstrated to stop the growth of Escherichia coli. A comparison of LpxC sequences from diverse bacteria identified 10 conserved His, Asp, and Glu residues that might play catalytic roles. Each amino acid was altered in both E. coli and Aquifex aeolicus LpxC and the catalytic activities of the variants were determined. Three His and one Asp residues (H79, H238, D246, and H265) are essential for catalysis based on the low activities (<0.1% of wild-type LpxC) of mutants with alanine substitutions at these positions. H79 and H238 likely coordinate zinc; the Zn(2+) content of the purified variant proteins is low and the specific activity is enhanced by the addition of Zn(2+). The third side chain to coordinate zinc is likely either H265 or D246 and a fourth ligand is likely a water molecule, as indicated by the hydroxamate inhibition, suggesting a His(3)H(2)O or His(2)AspH(2)O Zn(2+)-polyhedron in LpxC. The decreased zinc inhibition of LpxC mutants at E78 suggests that this side chain may coordinate a second, inhibitory Zn(2+) ion. Given the absence of any known Zn(2+) binding motifs, the active site of LpxC may have evolved differently than other well-studied zinc metalloamidases, a feature that should aid in the design of safe antibiotics.     

Characterization of Mycobacterium tuberculosis nicotinamidase/pyrazinamidase

The nicotinamidase/pyrazinamidase (PncA) of Mycobacterium tuberculosis is involved in the activation of the important front-line antituberculosis drug pyrazinamide by converting it into the active form, pyrazinoic acid. Mutations in the pncA gene cause pyrazinamide resistance in M. tuberculosis. The properties of M. tuberculosis PncA were characterized in this study. The enzyme was found to be a 20.89 kDa monomeric protein. The optimal pH and temperature of enzymatic activity were pH 7.0 and 40 degrees C, respectively. Inductively coupled plasma-optical emission spectrometry revealed that the enzyme was an Mn(2+)/Fe(2+)-containing protein with a molar ratio of [Mn(2+)] to [Fe(2+)] of 1 : 1; furthermore, the external addition of either type of metal ion had no apparent effect on the wild-type enzymatic activity. The activity of the purified enzyme was determined by HPLC, and it was shown that it possessed similar pyrazinamidase and nicotinamidase activity, by contrast with previous reports. Nine PncA mutants were generated by site-directed mutagenesis. Determination of the enzymatic activity and metal ion content suggested that Asp8, Lys96 and Cys138 were key residues for catalysis, and Asp49, His51, His57 and His71 were essential for metal ion binding. Our data show that M. tuberculosis PncA may bind metal ions in a manner different from that observed in the case of Pyrococcus horikoshii PncA.     

Porins of Pseudomonas fluorescens MFO as fibronectin-binding proteins

Gene araA encoding an L-arabinose isomerase (AraA) from the hyperthermophile, Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 496 residues with a calculated molecular mass of 56677 Da. The deduced amino acid sequence has 94.8% identical amino acids compared with the residues in a putative L-arabinose isomerase of Thermotoga maritima. The recombinant enzyme expressed in E. coli was purified to homogeneity by heat treatment, ion exchange chromatography and gel filtration. The thermophilic enzyme had a maximum activity of L-arabinose isomerization and D-galactose isomerization at 85 degrees C, and required divalent cations such as Co(2+) and Mn(2+) for its activity and thermostability. The apparent K(m) values of the enzyme for L-arabinose and D-galactose were 116 mM (v(max), 119 micromol min(-1) mg(-1)) and 250 mM (v(max), 14.3 micromol min(-1) mg(-1)), respectively, that were determined in the presence of both 1 mM Co(2+) and 1 mM Mn(2+). A 68% conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80 degrees C.     

Structure and mechanism of action of a novel phosphoglycerate mutase from Bacillus stearothermophilus

Bacillus stearothermophilus phosphoglycerate mutase (PGM), which interconverts 2- and 3-phosphoglyceric acid (PGA), does not require 2,3-diphosphoglyceric acid for activity. However, this enzyme does have an absolute and specific requirement for Mn(2+) ions for catalysis. Here we report the crystal structure of this enzyme complexed with 3PGA and manganese ions to 1.9 A resolution; this is the first crystal structure of a diphosphoglycerate-independent PGM to be determined. This information, plus the location of the two bound Mn(2+) ions and the 3PGA have allowed formulation of a possible catalytic mechanism for this PGM. In this mechanism Mn(2+) ions facilitate the transfer of the substrate's phosphate group to Ser62 to form a phosphoserine intermediate. In the subsequent phosphotransferase part of the reaction, the phosphate group is transferred from Ser62 to the O2 or O3 positions of the reoriented glycerate to yield the PGA product. Site-directed mutagenesis studies were used to confirm our mechanism and the involvement of specific enzyme residues in Mn(2+) binding and catalysis.     

Isolation and amino acid sequence of a peptide containing an epoxide-reactive residue from the thermolysin-digest of Scytalidium lignicolum acid protease B

Scytalidium lignicolum acid protease B, a pepstatin-insensitive acid protease, was modified by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) with the concomitant loss of its enzyme activity, and an EPNP-labeled peptide was isolated from the thermolysin-digest of the modified enzyme by HPLC. The amino acid sequence of the peptide was determined to be Ile-Leu-Glu-Thr-Gly, which corresponds to the sequence of residue Nos. 51-55 of the enzyme. The results of treatment of the labeled peptide with hydroxylamine suggested that the EPNP moiety is ester-linked to Glu53 of the enzyme. The amino acid sequence around Glu53 of the acid protease B showed high homology with those around the active site Asp residues of calf chymosin and porcine pepsin. These results show that it is highly possible that Glu53 of the acid protease B is one of the amino acid residues involved in its catalytic activity.