Characterization of bacterial acylpeptidyl-oligopeptidase.

Acylpeptidyl-oligopeptidase (AOP), which has been recently identified as a novel enzyme in a periodontopathic bacterium, Porphyromonas gingivalis, removes di- and tri-peptides from N-terminally acylated polypeptides, with a preference for hydrophobic P1-position amino acid residues. To validate enzymatic properties of AOP, characteristics of two bacterial orthologues from Bacteroides dorei (BdAOP), a Gram-negative intestinal rod, and Lysinibacillus sphaericus (LsAOP), a Gram-positive soil rod, were determined. Like P. gingivalis AOP (PgAOP), two orthologues more efficiently hydrolyzed N-terminal acylated peptidyl substrates than non-acylated ones. Optimal pH was shifted from 7.0 to 8.9 for N-acylated to 8.5-9.5 for non-acylated substrates, indicating preference for non-charged hydrophobic N-terminal residues. Hydrophobic P1- and P2-position preferences were common in the three AOPs, although PgAOP preferred Leu and the others preferred Phe at the P1 position. In vitro mutagenesis demonstrated that Phe in PgAOP was responsible for the P1 Leu preference. In addition, bacterial AOPs commonly liberated acetyl-Ser-Tyr from α-melanocyte-stimulating hormone. Taken together, although these three bacterial AOPs exhibited some variations in biochemical properties, the present study demonstrated the existence of a group of exopeptidases that preferentially liberates mainly dipeptides from N-terminally acylated polypeptides with a preference for hydrophobic P1 and P2-position residues.