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W2092056387 abstract "The α-amino acid ester hydrolase fromAcetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing the side chain peptide bond in β-lactam antibiotics. Data base searches revealed that the enzyme contains an active site serine consensus sequence Gly-X-Ser-Tyr-X-Gly that is also found in X-prolyl dipeptidyl aminopeptidase. The serine hydrolase inhibitorp-nitrophenyl-p′-guanidino-benzoate appeared to be an active site titrant and was used to label the α-amino acid ester hydrolase. Electrospray mass spectrometry and tandem mass spectrometry analysis of peptides from a CNBr digest of the labeled protein showed that Ser205, situated in the consensus sequence, becomes covalently modified by reaction with the inhibitor. Extended sequence analysis showed alignment of this Ser205with the catalytic nucleophile of some α/β-hydrolase fold enzymes, which posses a catalytic triad composed of a nucleophile, an acid, and a base. Based on the alignments, 10 amino acids were selected for site-directed mutagenesis (Arg85, Asp86, Tyr143, Ser156, Ser205, Tyr206, Asp338, His370, Asp509, and His610). Mutation of Ser205, Asp338, or His370 to an alanine almost fully inactivated the enzyme, whereas mutation of the other residues did not seriously affect the enzyme activity. Circular dichroism measurements showed that the inactivation was not caused by drastic changes in the tertiary structure. Therefore, we conclude that the catalytic domain of the α-amino acid ester hydrolase has an α/β-hydrolase fold structure with a catalytic triad of Ser205, Asp338, and His370. This distinguishes the α-amino acid ester hydrolase from the Ntn-hydrolase family of β-lactam antibiotic acylases. The α-amino acid ester hydrolase fromAcetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing the side chain peptide bond in β-lactam antibiotics. Data base searches revealed that the enzyme contains an active site serine consensus sequence Gly-X-Ser-Tyr-X-Gly that is also found in X-prolyl dipeptidyl aminopeptidase. The serine hydrolase inhibitorp-nitrophenyl-p′-guanidino-benzoate appeared to be an active site titrant and was used to label the α-amino acid ester hydrolase. Electrospray mass spectrometry and tandem mass spectrometry analysis of peptides from a CNBr digest of the labeled protein showed that Ser205, situated in the consensus sequence, becomes covalently modified by reaction with the inhibitor. Extended sequence analysis showed alignment of this Ser205with the catalytic nucleophile of some α/β-hydrolase fold enzymes, which posses a catalytic triad composed of a nucleophile, an acid, and a base. Based on the alignments, 10 amino acids were selected for site-directed mutagenesis (Arg85, Asp86, Tyr143, Ser156, Ser205, Tyr206, Asp338, His370, Asp509, and His610). Mutation of Ser205, Asp338, or His370 to an alanine almost fully inactivated the enzyme, whereas mutation of the other residues did not seriously affect the enzyme activity. Circular dichroism measurements showed that the inactivation was not caused by drastic changes in the tertiary structure. Therefore, we conclude that the catalytic domain of the α-amino acid ester hydrolase has an α/β-hydrolase fold structure with a catalytic triad of Ser205, Asp338, and His370. This distinguishes the α-amino acid ester hydrolase from the Ntn-hydrolase family of β-lactam antibiotic acylases. α-amino acid ester hydrolase p-nitrophenyl-p′-guanidino-benzoate p-nitrophenol d-2-nitro-5-[(phenylglycyl)amino]-benzoic acid electrospray mass spectrometry high-pressure liquid chromatography dimethylformamide Protein Data Bank The α-amino acid ester hydrolases have been known for their applicability in the biocatalytic synthesis of semisynthetic β-lactam antibiotics since 1972 (1Takahashi T. Yamazaki Y. Kato K. Isona M. J. Am. Chem. Soc. 1972; 94: 4035-4037Crossref PubMed Scopus (60) Google Scholar). These enzymes can hydrolyze the amide bond that connects the acyl side chain to the β-lactam nucleus. Starting from esterified acyl precursors, they can also catalyze the reverse reaction. Remarkable features of these enzymes are the ability to accept charged substrates such as α-amino acid esters, the preference for esters over amides, and the low pH optimum (pH 6.2) (2Takahashi T. Yamazaki Y. Kato K. Biochem. J. 1974; 137: 497-503Crossref PubMed Scopus (45) Google Scholar, 3Blinkovsky A.M. Markaryan A.N. Enzyme Microb. Technol. 1993; 15: 965-973Crossref PubMed Scopus (35) Google Scholar). Despite these attractive properties, a gene encoding an α-amino acid ester hydrolase (AEH)1 was only recently cloned and characterized (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (29) Google Scholar). Thus far, all the known β-lactam antibiotic acylases, such as penicillin G acylase (5Duggleby H.J. Tolley S.P. Hill C.P. Dodson E.J. Dodson G. Moody P.C.E. Nature. 1995; 373: 264-268Crossref PubMed Scopus (421) Google Scholar), penicillin V acylase (6Suresh C.G. Pundle A.V. Siva Raman H. Rao K.N. Brannigan J.A. McVey C.E. Verma C.S. Dauter Z. Dodson E.J. Dodson G.G. Nat. Struct. Biol. 1999; 6: 414-416Crossref PubMed Scopus (104) Google Scholar), and cephalosporin acylase (7Kim Y. Yoon K.-H. Khang Y. Turley S. Hol W.G.J. Structure. 2000; 8: 1059-1068Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), belong to the Ntn-hydrolase family. However, protein data base searches showed no homology of the AEH of Acetobacter turbidans with known β-lactam antibiotic acylases. The N-terminal amino acid sequence of the AEH was determined; it revealed a signal sequence, but no N-terminally located Thr, Ser, or Cys, characteristic for members of the Ntn-hydrolase family, was found. It was therefore postulated that the AEHs belong to a new class of β-lactam antibiotic acylases (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (29) Google Scholar).An alignment of the AEH sequence with those of homologous proteins showed the presence of the active site serine consensus motif GXSYXG (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (29) Google Scholar), which is described for the X-prolyl dipeptidyl aminopeptidases (8Chich J.-F. Chapot-Chartier M.-P. Ribadeau-Dumas B. Gripon J.-C. FEBS Lett. 1992; 314: 139-142Crossref PubMed Scopus (29) Google Scholar). No x-ray structure of the aminopeptidases is known, but they are members of a group of proteins that belong to the prolyl oligopeptidase family. Of this family two structures have been solved, which both contain an α/β-hydrolase fold (9Fülöp V. Böcskei Z. Polgár L. Cell. 1998; 94: 161-170Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 10Medrano F.J. Alonso J. Garcı́a J.L. Romero A. Bode W. Gomis-Rüth F.X. EMBO J. 1998; 17: 1-9Crossref PubMed Scopus (70) Google Scholar) and have a catalytic triad of Ser, Asp, and His. Therefore, it is possible that the X-prolyl dipeptidyl aminopeptidases and hence AEH also have a catalytic triad. This assumption is further supported by the identification of a catalytic triad in the recently solved crystal structure of a cocaine esterase (11Larsen N.A. Turner J.M. Stevens J. Rosser S.J. Basran A. Lerner R.A. Bruce N.C. Wilson I.A. Nat. Struct. Biol. 2001; 9: 17-21Crossref Scopus (102) Google Scholar) that is also related to AEH. Earlier experiments with inhibitors already suggested the importance of a histidine for the catalytic activity of AEH (12Ryu Y.W. Ryu D.D.Y. Enzyme Microb. Technol. 1988; 10: 239-245Crossref Scopus (12) Google Scholar). However, common serine hydrolase inhibitors such as phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, or Pefabloc SC showed no inhibition of AEH activity (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (29) Google Scholar, 12Ryu Y.W. Ryu D.D.Y. Enzyme Microb. Technol. 1988; 10: 239-245Crossref Scopus (12) Google Scholar). On the other hand, inhibition was observed with the serine hydrolase inhibitorp-nitrophenyl-p′-guanidino-benzoate (p-NPGB), but the inhibition was incomplete, which left uncertainty about the catalytic role of a serine in AEH (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (29) Google Scholar). In this study, we used active site labeling, site-directed mutagenesis, and sequence analysis to demonstrate that AEH is a member of a class of β-lactam antibiotic acylases that belongs to the α/β-hydrolase fold family and possesses a classical catalytic triad of Ser, Asp, and His. The α-amino acid ester hydrolases have been known for their applicability in the biocatalytic synthesis of semisynthetic β-lactam antibiotics since 1972 (1Takahashi T. Yamazaki Y. Kato K. Isona M. J. Am. Chem. Soc. 1972; 94: 4035-4037Crossref PubMed Scopus (60) Google Scholar). These enzymes can hydrolyze the amide bond that connects the acyl side chain to the β-lactam nucleus. Starting from esterified acyl precursors, they can also catalyze the reverse reaction. Remarkable features of these enzymes are the ability to accept charged substrates such as α-amino acid esters, the preference for esters over amides, and the low pH optimum (pH 6.2) (2Takahashi T. Yamazaki Y. Kato K. Biochem. J. 1974; 137: 497-503Crossref PubMed Scopus (45) Google Scholar, 3Blinkovsky A.M. Markaryan A.N. Enzyme Microb. Technol. 1993; 15: 965-973Crossref PubMed Scopus (35) Google Scholar). Despite these attractive properties, a gene encoding an α-amino acid ester hydrolase (AEH)1 was only recently cloned and characterized (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (29) Google Scholar). Thus far, all the known β-lactam antibiotic acylases, such as penicillin G acylase (5Duggleby H.J. Tolley S.P. Hill C.P. Dodson E.J. Dodson G. Moody P.C.E. Nature. 1995; 373: 264-268Crossref PubMed Scopus (421) Google Scholar), penicillin V acylase (6Suresh C.G. Pundle A.V. Siva Raman H. Rao K.N. Brannigan J.A. McVey C.E. Verma C.S. Dauter Z. Dodson E.J. Dodson G.G. Nat. Struct. Biol. 1999; 6: 414-416Crossref PubMed Scopus (104) Google Scholar), and cephalosporin acylase (7Kim Y. Yoon K.-H. Khang Y. Turley S. Hol W.G.J. Structure. 2000; 8: 1059-1068Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), belong to the Ntn-hydrolase family. However, protein data base searches showed no homology of the AEH of Acetobacter turbidans with known β-lactam antibiotic acylases. The N-terminal amino acid sequence of the AEH was determined; it revealed a signal sequence, but no N-terminally located Thr, Ser, or Cys, characteristic for members of the Ntn-hydrolase family, was found. It was therefore postulated that the AEHs belong to a new class of β-lactam antibiotic acylases (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (29) Google Scholar). An alignment of the AEH sequence with those of homologous proteins showed the presence of the active site serine consensus motif GXSYXG (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (29) Google Scholar), which is described for the X-prolyl dipeptidyl aminopeptidases (8Chich J.-F. Chapot-Chartier M.-P. Ribadeau-Dumas B. Gripon J.-C. FEBS Lett. 1992; 314: 139-142Crossref PubMed Scopus (29) Google Scholar). No x-ray structure of the aminopeptidases is known, but they are members of a group of proteins that belong to the prolyl oligopeptidase family. Of this family two structures have been solved, which both contain an α/β-hydrolase fold (9Fülöp V. Böcskei Z. Polgár L. Cell. 1998; 94: 161-170Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 10Medrano F.J. Alonso J. Garcı́a J.L. Romero A. Bode W. Gomis-Rüth F.X. EMBO J. 1998; 17: 1-9Crossref PubMed Scopus (70) Google Scholar) and have a catalytic triad of Ser, Asp, and His. Therefore, it is possible that the X-prolyl dipeptidyl aminopeptidases and hence AEH also have a catalytic triad. This assumption is further supported by the identification of a catalytic triad in the recently solved crystal structure of a cocaine esterase (11Larsen N.A. Turner J.M. Stevens J. Rosser S.J. Basran A. Lerner R.A. Bruce N.C. Wilson I.A. Nat. Struct. Biol. 2001; 9: 17-21Crossref Scopus (102) Google Scholar) that is also related to AEH. Earlier experiments with inhibitors already suggested the importance of a histidine for the catalytic activity of AEH (12Ryu Y.W. Ryu D.D.Y. Enzyme Microb. Technol. 1988; 10: 239-245Crossref Scopus (12) Google Scholar). However, common serine hydrolase inhibitors such as phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, or Pefabloc SC showed no inhibition of AEH activity (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (29) Google Scholar, 12Ryu Y.W. Ryu D.D.Y. Enzyme Microb. Technol. 1988; 10: 239-245Crossref Scopus (12) Google Scholar). On the other hand, inhibition was observed with the serine hydrolase inhibitorp-nitrophenyl-p′-guanidino-benzoate (p-NPGB), but the inhibition was incomplete, which left uncertainty about the catalytic role of a serine in AEH (4Polderman-Tijmes J.J. Jekel P.A. van Merode A. Floris T.A.G. van de Laan J.-M. Sonke T. Janssen D.B. Appl. Environ. Microbiol. 2002; 68: 211-218Crossref PubMed Scopus (29) Google Scholar). In this study, we used active site labeling, site-directed mutagenesis, and sequence analysis to demonstrate that AEH is a member of a class of β-lactam antibiotic acylases that belongs to the α/β-hydrolase fold family and possesses a classical catalytic triad of Ser, Asp, and His. We thank Dr. P. Terpstra for sequencing the material presented in this study." @default.
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W2092056387 title "Identification of the Catalytic Residues of α-Amino Acid Ester Hydrolase from Acetobacter turbidans by Labeling and Site-directed Mutagenesis" @default.
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W2092056387 doi "https://doi.org/10.1074/jbc.m204143200" @default.
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