Identification of the catalytic residues of alpha-amino acid ester hydrolase from Acetobacter turbidans by labeling and site-directed mutagenesis.
The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing the side chain peptide bond in beta-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 alpha-amino acid ester hydrolase. Electrospray mass spectrometry and tandem mass spectrometry analysis of peptides from a CNBr digest of the labeled protein showed that Ser(205), situated in the consensus sequence, becomes covalently modified by reaction with the inhibitor. Extended sequence analysis showed alignment of this Ser(205) with the catalytic nucleophile of some alpha/beta-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 (Arg(85), Asp(86), Tyr(143), Ser(156), Ser(205), Tyr(206), Asp(338), His(370), Asp(509), and His(610)). Mutation of Ser(205), Asp(338,) or His(370) 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 alpha-amino acid ester hydrolase has an alpha/beta-hydrolase fold structure with a catalytic triad of Ser(205), Asp(338), and His(370). This distinguishes the alpha-amino acid ester hydrolase from the Ntn-hydrolase family of beta-lactam antibiotic acylases.
Polderman-Tijmes
Jolanda J
JJ
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, NL-9747 AG Groningen, The Netherlands.
Jekel
Peter A
PA
Jeronimus-Stratingh
C Margot
CM
Bruins
Andries P
AP
Van Der Laan
Jan-Metske
JM
Sonke
Theo
T
Janssen
Dick B
DB
D000091:Acetobacter / Q000201:enzymology*; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D007769:Lactams; D008715:Methionine / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013912:Threonine / Q000737:chemistry; D013997:Time Factors
D000900:Anti-Bacterial Agents; D007769:Lactams; D010455:Peptides; D013912:Threonine; D012694:Serine; D008715:Methionine; D002265:Carboxylic Ester Hydrolases; C058058:alpha-amino acid esterase