A Secreted Aminopeptidase of Pseudomonas aeruginosa

Abstract

Using leucine-p-nitroanilide (Leu-pNA) as a sub- strate, we demonstrated aminopeptidase activity in the culture filtrates of several Pseudomonas aerugi- nosa strains. The aminopeptidase was partially puri- fied by DEAE-cellulose chromatography and found to be heat stable. The apparent molecular mass of the enzyme was ?56 kDa; hence, it was designated AP56. Heating (70 °C) of the partially purified aminopepti- dase preparations led to the conversion of AP56 to a ?28-kDa protein (AP28) that retained enzyme activity, a reaction that depended on elastase (LasB). The pH optimum for Leu-pNA hydrolysis by AP28 was 8.5. This activity was inhibited by Zn chelators but not by in- hibitors of serine- or thiol-proteases, suggesting that AP28 is a Zn-dependent enzyme. Of several amino acid p-nitroanilide derivatives examined, Leu-pNA was the preferred substrate. The sequences of the first 20 res- idues of AP56 and AP28 were determined. A search of the P. aeruginosa genomic data base revealed a perfect match of these sequences with positions 39–58 and 273–291, respectively, in a 536-amino acid residue open reading frame predicted to encode an aminopeptidase. A search for sequence similarities with other proteins revealed 52% identity with Streptomyces griseus amin- opeptidase, ?35% identity with Saccharomyces cerevi- siae aminopeptidase Y and a hypothetical aminopepti- dase from Bacillus subtilis, and 29–32% with Aeromonas caviae, Vibrio proteolyticus, and Vibrio cholerae aminopeptidases. The residues potentially in- volved in zinc coordination were conserved in all these proteins. Thus, P. aeruginosa aminopeptidase may be- long to the same family (M28) of metalloproteases.