| Protease IV is a lysine-specific endopeptidase of Pseudomonas aeruginosa. In the present study, the protease IV gene was cloned and analyzed for post-translational processing, the role in virulence and the amino acids comprising the active site of this enzyme. The enzyme was expressed in a protease IV-negative Pseudomonas species, Pseudomonas putida. The enzyme gene product was processed into an active form in P. putida as determined by the N-terminal amino acid sequence, molecular mass, protease activity, substrate specificity and inhibitor reactivity. Expression of the cloned protease IV gene in P. putida resulted in enzyme activity significantly greater (∼5-fold) than in P. aeruginosa. Immunoblotting of P. putida cell extracts revealed a protein with the same molecular mass as the mature protease and two polypeptides, one representing the entire gene product (∼48 kDa) and the other lacking the signal sequence (∼45 kDa). When intrastromally injected into rabbit corneas, P. putida producing protease IV, relative to P. putida with the vector alone, caused a three-fold increase in ocular inflammation and tissue damage. Based on amino acid sequence homology of protease IV and other serine proteases, we hypothesized that His-72, Asp-122 and Ser-198, likely constituents of the catalytic triad, contributed to lysine-specific activity of protease IV. In addition to the catalytic triad, identical amino acid residues including His-116, Ser-197 and Ser-200 located close to the triad were investigated. Mutants with alanine replacement at amino acid His-72, Asp-122, Ser-197 or Ser-198 retained no proteolytic activity and there was an accumulation of inactive forms of protease IV. In contrast, His-116 or Ser-200 mutants possessed high protease activity and produced the mature protease IV. In summary, the protease IV gene was successfully cloned and expressed in a new host species. The protease IV is synthesized as a large precursor that is processed intracellularly and secreted into the extracellular milieu as a mature protease. A significant correlation between protease IV production and corneal virulence was confirmed using the protease IV construct in P. putida. Additionally, the results suggest that the catalytic triad and Ser-197 are critical amino acid residues for the proper processing and activity of protease IV. |
