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 intramolecular chaperone required for the folding of the enzyme and as an inhibitor that prevents activity of the enzyme before its secretion into the extracellular medium. Since expression of the lasB gene, which encodes elastase, in Pseudomonas putida did not result in extracellular elastase activity, it has been suggested that the enzyme is not recognized by the Xcp secretion machinery of the heterologous host. Here, it is demonstrated that the proenzyme is normally processed in P. putida and that it is indeed not actively secreted by the Xcp machinery. Nevertheless, substantial amounts of the enzyme were detected in the extracellular medium. Co-immunoprecipitations revealed that the extracellular enzyme was associated with the propeptide, which explains the lack of enzymic activity. Since the propeptide-enzyme complex in P. putida apparently does not dissociate spontaneously, it is concluded 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, respectively, of the propeptide (where position +1 is defined as the first residue of the mature enzyme) destabilized the propeptide-enzyme complex. It is concluded that Ala-15 and Thr-153 are required for the inhibitor function, but not for the chaperone function of the propeptide.
Braun
Peter
P
Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands1.
Bitter
Wilbert
W
Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands1.
Tommassen
Jan
J
Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands1.
D015153:Blotting, Western / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010196:Pancreatic Elastase / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011233:Precipitin Tests / Q000379:methods; D011485:Protein Binding; D017510:Protein Folding; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D014169:Transformation, Bacterial / Q000235:genetics
D004792:Enzyme Precursors; D010196:Pancreatic Elastase