A DmpA‐homologous protein from Pseudomonas sp. is a dipeptidase specific for β‐alanyl dipeptides

Abstract

Many different kinds of microbial hydrolases acting d-stereoselectively on amino acid amides or peptides have been characterized, and some of them have been applied to the production of optically active d-amino acids from the corresponding racemic amino acid amides [1]. The d-stereoselective amidases and peptidases known to date can be classified into four groups based on their primary structures. 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) A recombinant BapA exhibiting hydrolytic activity toward d-alaninep-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 9.0-10.0, 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 b-alanyl dipeptides including b-Ala-l-Ala, b-Ala-Gly, b-Ala-l-His (carnosine), b-Ala-l-Leu, and (b-Ala) 2 with high efficiency compared to d-alanine-p-nitroanilide. b-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 b-Ala-Xaa dipeptidase.

Amidases: versatile enzymes in nature

“…The DmpA also known as L-aminopeptidase D-Ala-esterase/amidase, hydrolyzed D-configuration of the alanine substrates (e.g. alaninep-nitroanilide, alaninamide, and alanine methylester) while the enzyme acted as an L-stereoselective aminopeptidase on tripeptide Ala-(Gly) 2 substrate thus indicating a reverse stereoselectivity (Komeda et al 2004;Komeda and Asano 2005).…”

Hydrolysis of Amides

“…A broad evaluation of the substrate spectrum of a homologous enzyme from Pseudomonas sp. MCI3434 (BapA, 43% sequence identity), a few years later, revealed that dipeptides with a b-alanine residue at their amino terminus as well as b-alanine amide were hydrolyzed much more efficiently than D-alanine-p-nitroanilide [82]. BapA was thus named b-Ala-Xaa dipeptidase instead of L-aminopeptidase D-alanine-esterase/amidase.…”

“…Most of these methods are chemical in nature [319][320][321], but enzymatic methods applying, for instance, a lipase [322,323], aminoacylase [324], penicillin G acylase [325], peptide deformylase [9], and aminomutase [326] have also been described (for a review see Reference [327]). An enzyme with activity toward a b-amino acid amide was described in 2005 by Komeda and Asano [82]. This enzyme was purified from Pseudomonas sp.…”

“…Much higher activities were observed for dipeptides containing b-alanine (also called b-homoglycine (H-b-hGly-OH)) at their N-terminus, including b-Ala-L-Ala, b-Ala-Gly, b-Ala-L-His (L-carnosine), b-Ala-L-Leu and (b-Ala) 2 , and b-Ala-NH 2 . BapA was therefore called b-Ala-Xaa dipeptidase (EC 3.4.13.-) [82]. The enantioselectivity of BapA for b-amino acid amides has not been reported yet.…”

Biodegradable Polymers: Recent Developments and New Perspectives

“…PahZ1 enzymes are classified into the α/β-hydrolase_5 family, which includes putative PHB depolymerase (LpqC) from Bordetella parapertussis, based on the ESTHER database (http://bioweb.ensam.inra.fr/esther). As shown in Figure 10, the residues composing their catalytic triads, in which the Ser residue formed together with Asp and His residues, are conserved in PahZ1 KT In addition to the PAA-hydrolysing enzymes (PahZ1 KT-1 , PahZ2 KT-1 , and PahZ1 KP-2 ), five β-aminopeptidases (three BapA enzymes, one BapF enzyme, and one DmpA enzyme) hydrolyse short β-peptides and β-amino-acid containing peptides [135][136][137][138][139][140][141][142]. Their properties are listed in Table 2.…”