The DmpA aminopeptidase from Ochrobactrum anthropi LMG7991 is the prototype of a new terminal nucleophile hydrolase family

Abstract

The DmpA (D-aminopeptidase A) protein produced by Ochrobactrum anthropi hydrolyses p-nitroanilide derivatives of glycine and D-alanine more efficiently than that of L-alanine. When regular peptides are utilized as substrates, the enzyme behaves as an aminopeptidase with a preference for N-terminal residues in an L configuration, thus exemplifying an interesting case of stereospecificity reversal. The best-hydrolysed substrate is L-Ala-Gly-Gly, but tetra- and penta-peptides are also efficiently hydrolysed. The gene encodes a 375-residue precursor, but the active enzyme contains two polypeptides corresponding to residues 2-249 (α-subunit) and 250-375 (β-subunit) of the precursor. Residues 249 and 250 are a Gly and a Ser respectively, and various substitutions performed by site-directed mutagenesis result in the production of an uncleaved and inactive protein. The N-terminal Ser residue of the β-subunit is followed by a hydrophobic peptide, which is predicted to form a β-strand structure. All these properties strongly suggest that DmpA is an N-terminal amidohydrolase. An exploration of the databases highlights the presence of a number of open reading frames encoding related proteins in various bacterial genomes. Thus DmpA is very probably the prototype of an original family of N-terminal hydrolases.