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 (alpha-subunit) and 250-375 (beta-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 beta-subunit is followed by a hydrophobic peptide, which is predicted to form a beta-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.

A Novel β-Peptidyl Aminopeptidase (BapA) from Strain 3-2W4 Cleaves Peptide Bonds of Synthetic β-Tri- and β-Dipeptides

“…The results of our inhibition studies revealed that BapA was inhibited neither by the protease inhibitors phenylmethylsulfonyl fluoride and EDTA nor by divalent metal ions and reducing agents. No inhibitor has been found so far for DmpA from O. anthropi (9). BapA remains active even in the presence of 50% (vol/vol) ethylene glycol during purification.…”

“…Therefore, we named the enzyme BapA for ␤-peptidyl aminopeptidase. DmpA from O. anthropi is described as the prototype of a new family of Ntn hydrolases (23), which are activated by a self-catalyzed protein splicing process between two conserved residues to open access to the catalytic N-terminal residue (serine, threonine, or cysteine) (9). The two subunits of DmpA from O. anthropi have molecular masses of 26.6 and 13.7 kDa, respectively; this corresponds well with the molecular masses of the BapA subunits.…”

6-Aminohexanoate Oligomer Hydrolases from the Alkalophilic Bacteria Agromyces sp. Strain KY5R and Kocuria sp. Strain KY2

“…The 10 N-terminal residues obtained by protein sequencing were identical to those deduced from the nucleotide sequence starting from Thr267, demonstrating that NylC A and NylC K were subjected to specific cleavage at Asn266/Thr267, which has previously been identified as a cleavage site in NylC p2 (4). The observed processing is a specific feature of the N-terminal nucleophile (Ntn) hydrolase family, in which cleavage is performed auto-catalytically to generate two subunits (1,2,3,8).…”

Three-dimensional Structure of Nylon Hydrolase and Mechanism of Nylon-6 Hydrolysis

“…DmpA is an aminopeptidase that hydrolyzes peptides, with a preference for N-terminal residues in an L-configuration (L-Ala-Gly-Gly) and also amide or ester derivatives of D-Ala (19). BapA hydrolyzes ␤-oligopeptides and mixed ␤/␣-oligopeptides with a ␤-amino acid residue at the N terminus (20).…”