A DmpA-homologous protein from Pseudomonas sp is a dipeptidase specific for beta-alanyl dipeptides

作者：Komeda H; Asano Y^*

来源：FEBS Journal, 2005, 272(12): 3075-3084.

DOI：10.1111/j.1742-4658.2005.04721.x

• amidase
• alanine
• dipeptidase
• DmpA
• Pseudomonas sp.

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 & 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 (alpha-peptide) and 239-366 (beta-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 degrees C and pH 9.0-10.0, 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 than L-alanine-p-nitroanilide the same as DmpA. Furthermore, BapA was found to hydrolyze peptide bonds of beta-alanyl dipeptides including beta-Ala-L-Ala, beta-Ala-Gly, beta-Ala-L-His (carnosine), beta-Ala-L-Leu, and (beta-Ala)(2) with high efficiency compared to D-alanine-p-nitroanitide. beta-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 beta-Ala-Xaa dipeptidase.

• 出版日期2005-6