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We have determined the nucleotide sequence of a DNA fragment covering the flanking region of the R-stereoselective amidase gene, ramA, from the Pseudomonassp. MCI3434 genome and found an additional gene, bapA, coding for a protein showing sequence similarity to DmpA aminopeptidase from Ochrobactrum anthropiLMG7991 (43% identity). | ềFEBS Journal A DmpA-homologous protein from Pseudomonas sp. is a dipeptidase specific for 0-alanyl dipeptides Hidenobu Komeda and Yasuhisa Asano Biotechnology Research Center Toyama PrefecturalUniversity Toyama Japan Keywords amidase b-alanine dipeptidase DmpA Pseudomonas sp. Correspondence Y. Asano Biotechnology Research Center Toyama PrefecturalUniversity 5180 Kurokawa Kosugi Toyama 939-0398 Japan Fax 81 766 562498 Tel 81 766 567500 E-mail asano@pu-toyama.ac.jp Received 18 March 2005 revised 12 April 2005 accepted 18 April 2005 doi 10.1111 j.1742-4658.2005.04721.x We have determined the nucleotide sequence of a DNA fragment covering the flanking region of the R-stereoselective amidase gene ramA from the Pseudomonas sp. MCI3434 genome and found an additional gene bapA coding for a protein showing sequence similarity to DmpA aminopeptidase from Ochrobactrum anthropi LMG7991 43 identity . The DmpA called L-aminopeptidase D-Ala-esterase amidase hydrolyzes alanine-p-nitroani-lide alaninamide and alanine methylester with a preference for the D-con-figuration of the alanine whereas the enzyme acts as an L-stereoselective aminopeptidase on a tripeptide Ala- Gly 2 indicating a reverse stereoselectivity Fanuel L Goffin C Cheggour A Devreese B Van Driessche G Joris B Van Beeumen J Frere J-M 1999 Biochem J 341 147-155 . A recombinant BapA exhibiting hydrolytic activity toward D-alanine-p-nitroanilide was purified from the cell-free extract of an Escherichia coli transformant overexpressing the bapA gene and characterized. The purified enzyme contained two polypeptides corresponding to residues 1-238 a-peptide and 239-366 b-peptide of the precursor as observed for DmpA. On gel-filtration chromatography BapA in the native form appeared to be a tetramer. It had maximal activity at 60 C and pH 9.010.0 and was inactivated in the presence of p-chloromercuribenzoate N-ethylmaleimide dithiothreitol Zn2 Ag Cd2 or Hg2 . The enzyme hydrolyzed D-alanine-p-nitroanilide more efficiently
