This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Polderman-Tijmes, J. J. Articles by Janssen, D. B. Search for Related Content PubMed PubMed Citation Articles by Polderman-Tijmes, J. J. Articles by Janssen, D. B. Agricola Articles by Polderman-Tijmes, J. J. Articles by Janssen, D. B.

Cloning, Sequence Analysis, and Expression in Escherichia coli of the Gene Encoding an -Amino Acid Ester Hydrolase from Acetobacter turbidans

Jolanda J. Polderman-Tijmes,¹ Peter A. Jekel,¹ Erik J. de Vries,¹ Annet E. J. van Merode,¹ René Floris,^,1 Jan-Metske van der Laan,² Theo Sonke,³ and Dick B. Janssen^1*

Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, NL-9747 AG Groningen,¹ DSM Food Specialties, 2600 MA, Delft,² DSM Research, 6160 MD Geleen, The Netherlands³

Received 9 August 2001/ Accepted 2 November 2001

The -amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing ß-lactam antibiotics, such as cephalexin and ampicillin. N-terminal amino acid sequencing of the purified -amino acid ester hydrolase allowed cloning and genetic characterization of the corresponding gene from an A. turbidans genomic library. The gene, designated aehA, encodes a polypeptide with a molecular weight of 72,000. Comparison of the determined N-terminal sequence and the deduced amino acid sequence indicated the presence of an N-terminal leader sequence of 40 amino acids. The aehA gene was subcloned in the pET9 expression plasmid and expressed in Escherichia coli. The recombinant protein was purified and found to be dimeric with subunits of 70 kDa. A sequence similarity search revealed 26% identity with a glutaryl 7-ACA acylase precursor from Bacillus laterosporus, but no homology was found with other known penicillin or cephalosporin acylases. There was some similarity to serine proteases, including the conservation of the active site motif, GXSYXG. Together with database searches, this suggested that the -amino acid ester hydrolase is a ß-lactam antibiotic acylase that belongs to a class of hydrolases that is different from the Ntn hydrolase superfamily to which the well-characterized penicillin acylase from E. coli belongs. The -amino acid ester hydrolase of A. turbidans represents a subclass of this new class of ß-lactam antibiotic acylases.

Present address: NIZO, 6710 BA, Ede, The Netherlands.

This article has been cited by other articles:

• Barends, T. R. M., Polderman-Tijmes, J. J., Jekel, P. A., Williams, C., Wybenga, G., Janssen, D. B., Dijkstra, B. W. (2006). Acetobacter turbidans {alpha}-Amino Acid Ester Hydrolase: HOW A SINGLE MUTATION IMPROVES AN ANTIBIOTIC-PRODUCING ENZYME. J. Biol. Chem. 281: 5804-5810 [Abstract] [Full Text]  
• Barends, T. R. M., Polderman-Tijmes, J. J., Jekel, P. A., Hensgens, C. M. H., de Vries, E. J., Janssen, D. B., Dijkstra, B. W. (2003). The Sequence and Crystal Structure of the {alpha}-Amino Acid Ester Hydrolase from Xanthomonas citri Define a New Family of {beta}-Lactam Antibiotic Acylases. J. Biol. Chem. 278: 23076-23084 [Abstract] [Full Text]  
• Polderman-Tijmes, J. J., Jekel, P. A., Jeronimus-Stratingh, C. M., Bruins, A. P., van der Laan, J.-M., Sonke, T., Janssen, D. B. (2002). Identification of the Catalytic Residues of alpha -Amino Acid Ester Hydrolase from Acetobacter turbidans by Labeling and Site-directed Mutagenesis. J. Biol. Chem. 277: 28474-28482 [Abstract] [Full Text]