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

Rivka Cahan, Itschak Axelrad, Mary Safrin, Dennis E. Ohman, Efrat Kessler*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Using leucine-p-nitroanilide (Leu-pNA) as a substrate, we demonstrated aminopeptidase activity in the culture filtrates of several Pseudomonas aeruginosa strains. The aminopeptidase was partially purified 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 aminopeptidase 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 inhibitors 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 residues 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 aminopeptidase, ∼35% identity with Saccharomyces cerevisiae aminopeptidase Y and a hypothetical aminopeptidase from Bacillus subtilis, and 29-32% with Aeromonas caviae, Vibrio proteolyticus, and Vibrio cholerae aminopeptidases. The residues potentially involved in zinc coordination were conserved in all these proteins. Thus, P. aeruginosa aminopeptidase may belong to the same family (M28) of metalloproteases.

Original languageEnglish
Pages (from-to)43645-43652
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number47
DOIs
StatePublished - 23 Nov 2001

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