A new variant of the Ntn hydrolase fold revealed by the crystal structure of l-aminopeptidase d-Ala-esterase/amidase from Ochrobactrum anthropi

Abstract

Background: The l-aminopeptidase d-Ala-esterase/amidase from Ochrobactrum anthropi (DmpA) releases the N-terminal l and/or d-Ala residues from peptide substrates. This is the only known enzyme to liberate N-terminal amino acids with both d and l stereospecificity. The DmpA active form is an αβ heterodimer, which results from a putative autocatalytic cleavage of an inactive precursor polypeptide. Results: The crystal structure of the enzyme has been determined to 1.82 Å resolution using the multiple isomorphous replacement method. The heterodimer folds into a single domain organised as an αββα sandwich in which two mixed β sheets are flanked on both sides by two α helices. Conclusions: DmpA shows no similarity to other known aminopeptidases in either fold or catalytic mechanism, and thus represents the first example of a novel family of aminopeptidases. The protein fold of DmpA does, however, show structural homology to members of the N-terminal nucleophile (Ntn) hydrolase superfamily. DmpA presents functionally equivalent residues in the catalytic centre when compared with other Ntn hydrolases, and is therefore likely to use the same catalytic mechanism. In spite of this homology, the direction and connectivity of the secondary structure elements differ significantly from the consensus Ntn hydrolase topology. The DmpA structure thus characterises a new subfamily, but supports the common catalytic mechanism for these enzymes suggesting an evolutionary relationship.