A secreted aminopeptidase of Pseudomonas aeruginosa - Identification, primary structure, and relationship to other aminopeptidases

Using leucine-p-nitroanilide (Leu-pNA) as a substrate, we demonstrated amin opeptidase activity in the culture filtrates of several Pseudomonas aerugin osa strains. The aminopeptidase was partially purified by DEAE-cellulose ch romatography and found to be heat stable. The apparent molecular mass of th e enzyme was similar to 56 kDa; hence, it was designated AP(56). Heating (7 0 degreesC) of the partially purified aminopeptidase preparations led to th e conversion of AP56 to a similar to 28-kDa protein (AP(28)) that retained enzyme activity, a reaction that depended on elastase (LasB). The pH optimu m for Leu-pNA hydrolysis by AP(28) was 8.5. This activity was inhibited by Zn chelators but not by inhibitors of serine- or thiol-proteases, suggestin g that AP(28) is a Zn-dependent enzyme. Of several amino acid p-nitroanilid e derivatives examined, Leu-pNA was the preferred substrate. The sequences of the first 20 residues of AP(56), and AP(28) were determined. A search of the P. aeruginosa genomic data base revealed a perfect match of these sequ ences with positions 39-58 and 273-291, respectively, in a 536-amino acid r esidue open reading frame predicted to encode an aminopeptidase. A search f or sequence similarities with other proteins revealed 52% identity with Str eptomyces griseus aminopeptidase, similar to 35% identity with Saccharomyce s cerevisiae aminopeptidase Y and a hypothetical aminopeptidase from Bacill us subtilis, and 29-32% with Aeromonas caviae, Vibrio proteolyticus, and Vi brio cholerae aminopeptidases. The residues potentially involved in zinc co ordination were conserved in all these proteins. Thus, P. aeruginosa aminop eptidase may belong to the same family (M28) of metalloproteases.