A DmpA-homologous protein from Pseudomonas sp. is a dipeptidase specific for beta-alanyl dipeptides.
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 J341, 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-nitroanilide. 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.
Komeda
Hidenobu
H
Biotechnology Research Center, Toyama Prefectural University, Kosugi, Toyama, Japan.
Asano
Yasuhisa
Y
D000409:Alanine / Q000378:metabolism*; D000581:Amidohydrolases / Q000235:genetics; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D021901:DNA, Intergenic; D004150:Dipeptidases / Q000235:genetics / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D021901:DNA, Intergenic; D004151:Dipeptides; D004791:Enzyme Inhibitors; D007477:Ions; D008670:Metals; D000626:Aminopeptidases; C118321:DmpA protein, Ochrobactrum anthropi; D004150:Dipeptidases; D000581:Amidohydrolases; C019665:amidase; D000409:Alanine