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J. Biol. Chem., Vol. 277, Issue 32, 28474-28482, August 9, 2002

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Identification of the Catalytic Residues of -Amino Acid Ester Hydrolase from Acetobacter turbidans by Labeling and Site-directed Mutagenesis^*

Jolanda J. Polderman-Tijmes, Peter A. Jekel, C. Margot Jeronimus-Stratingh^§, Andries P. Bruins^§, Jan-Metske van der Laan^¶, Theo Sonke, and Dick B. Janssen^**

From the  Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, NL-9747 AG Groningen, The Netherlands, ^§ Mass Spectrometry Core Facility, University of Groningen, A. Deusinglaan 1, 9716 AV Groningen, The Netherlands, ^¶ DSM Food Specialties, P. O. Box 1, 2600 MA Delft, The Netherlands, and  DSM Research, P. O. Box 16, 6160 MD Geleen, The Netherlands

The -amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing the side chain peptide bond in -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 inhibitor p-nitrophenyl-p'-guanidino-benzoate appeared to be an active site titrant and was used to label the -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 Ser²⁰⁵ with the catalytic nucleophile of some /-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, Asp^338, 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 -amino acid ester hydrolase has an /-hydrolase fold structure with a catalytic triad of Ser²⁰⁵, Asp³³⁸, and His³⁷⁰. This distinguishes the -amino acid ester hydrolase from the Ntn-hydrolase family of -lactam antibiotic acylases.

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