Activation of Pseudomonas aeruginosa elastase in Pseudomonas putida by triggering dissociation of the propeptide-enzyme complex

The propeptide of Pseudomonas aeruginosa elastase functions both as an intr amolecular chaperone required for the folding of the enzyme and as an inhib itor that prevents activity of the enzyme before its secretion into the ext racellular medium. Since expression of the lasB gene, which encodes elastas e, in Pseudomonas putida did not result in extracellular elastase activity, it has been suggested that the enzyme is not recognized by the Xcp secreti on machinery of the heterologous host. Here, it is demonstrated that the pr oenzyme is normally processed in P. putida and that it is indeed not active ly secreted by the Xcp machinery. Nevertheless, substantial amounts of the enzyme were detected in the extracellular medium. Co-immunoprecipitations r evealed that the extracellular enzyme was associated with the propeptide, w hich explains the lack of enzymic activity. Since the propeptide-enzyme com plex in P. putida apparently does not dissociate spontaneously, it is concl uded that a host-specific factor is required to induce this event. Mutants were selected which showed extracellular elastase activity. Two mutations, located within the lasB gene, were further characterized. These mutations, resulting in the substitution of Ala and Thr at positions -15 and -153, res pectively, of the propeptide (where position +1 is defined as the first res idue of the mature enzyme) destabilized the propeptide-enzyme complex. It i s concluded that Ala-15 and Thr-153 are required for the inhibitor function , but not for the chaperone function of the propeptide.