Alkaline Proteinase Inhibitor of Pseudomonas aeruginosa

Abstract

Alkaline proteinase inhibitor of Pseudomonas aeruginosa is a 11.5-kDa, high affinity inhibitor of the serralysin class of zinc-dependent proteinases secreted by several Gram-negative bacteria. X-ray crystallography of the proteinase-inhibitor complex reveals that five Nterminal inhibitor residues occupy the extended substrate binding site of the enzyme and that the catalytic zinc is chelated by the ␣-amino and carbonyl groups of the N-terminal residue of the inhibitor. In this study, we assessed the effect of alteration of inhibitor residues 2-5 on its affinity for Pseudomonas alkaline proteinase (APR) as derived from the ratio of the dissociation and associate rate constants for formation of the enzymeinhibitor complex. The largest effect was observed at position Ser-2, which occupies the S1 pocket of the enzyme and donates a hydrogen bond to the carboxyl group of the catalytic Glu-177 of the proteinase. Substitution of Asp, Arg, or Trp at this position increased the dissociation constant K D by 35-, 180-, and 13-fold, respectively. Mutation at positions 3-5 of the trunk also resulted in a reduction in enzyme-inhibitor affinity, with the exception of an I4W mutant, which exhibited a 3-fold increase in affinity. Molecular dynamics simulation of the complex formation between the catalytic domain of APR and the S2D mutant showed that the carboxyl of Asp-2 interacts with the catalytic zinc, thereby partially neutralizing the negative charge that otherwise would clash with the carboxyl group of Glu-177 of APR. Simulation of the interaction between the alkaline proteinase and the I4W mutant revealed a major shift in the loop comprised of residues 189 -200 of the enzyme that allowed formation of a stacking interaction between the aromatic rings of Ile-4 of the inhibitor and Tyr-158 of the proteinase. This new interaction could account for the observed increase in enzyme-inhibitor affinity.

Downstream processing, characterization, and structure–function relationship of solvent‐, detergent‐, psychro‐, thermo‐, alkalistable metalloprotease from metal‐, solvent‐tolerant psychrotrophic Pseudomonas putida SKG‐1 isolate

“…aeruginosa are known to produce at least four different kinds of proteases, of which metalloproteases of ∼33 kDa (also termed as pseudolysin) and alkaline protease of ∼50 kDa are predominant,18 and our alkaline protease appears quite similar to ∼50 kDa alkaline proteases 18, 19. Feltzer et al20 stated that Pseudomonas , Serratia and Erwinia species secrete homologous ∼50 kDa alkaline proteinases which belong to the metzincin (Zn 2+ ) metalloproteinase superfamily.…”