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A DmpA‐homologous protein from Pseudomonas sp. is a dipeptidase specific for β‐alanyl dipeptides
Author(s) -
Komeda Hidenobu,
Asano Yasuhisa
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04721.x
Subject(s) - aminopeptidase , chemistry , alanine , stereochemistry , peptide , esterase , stereoselectivity , enzyme , biochemistry , microbiology and biotechnology , amino acid , biology , leucine , catalysis
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 ‐nitroanilide, alaninamide, and alanine methylester with a preference for the d ‐configuration 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 & Frère 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 (α‐peptide) and 239–366 (β‐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 9.0–10.0, and was inactivated in the presence of p ‐chloromercuribenzoate, N ‐ethylmaleimide, dithiothreitol, Zn 2+ , Ag + , Cd 2+ or Hg 2+ . The enzyme hydrolyzed d ‐alanine ‐p ‐nitroanilide more efficiently than l ‐alanine‐ p ‐nitroanilide the same as DmpA. Furthermore, BapA was found to hydrolyze peptide bonds of β‐alanyl dipeptides including β‐Ala‐ l ‐Ala, β‐Ala‐Gly, β‐Ala‐ l ‐His (carnosine), β‐Ala‐ l ‐Leu, and (β‐Ala) 2 with high efficiency compared to d ‐alanine‐ p ‐nitroanilide. β‐Alaninamide was also efficiently hydrolyzed, but the enzyme did not act on the peptides containing proteinogenic amino acids or their d ‐counterparts for N‐terminal residues. Based on its unique substrate specificity, the enzyme should not be called l ‐aminopeptidase d ‐Ala‐esterase/amidase but β‐Ala‐Xaa dipeptidase.