This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kawamoto, S Articles by Okuda, K Search for Related Content PubMed PubMed Citation Articles by Kawamoto, S Articles by Okuda, K

Site-directed mutagenesis of Glu-141 and His-223 in Pseudomonas aeruginosa elastase: catalytic activity, processing, and protective activity of the elastase against Pseudomonas infection.

Department of Bacteriology, Yokohama City University School of Medicine, Japan.

ABSTRACT

Both Pseudomonas aeruginosa elastase and Bacillus thermoproteolyticus thermolysin are zinc metalloproteases. On the basis of the high homology of the P. aeruginosa elastase with the Bacillus thermolysin, we hypothesized that Glu-141 and His-223 are the key residues for catalytic activity of the Pseudomonas elastase. To test this possibility, we replaced Glu-141 with Asp, Gln, and Gly and His-223 with Gly, Glu, and Leu by site-directed mutagenesis. These substitutions dramatically diminished the proteolytic activities of the mutant elastases when they were expressed in Escherichia coli cells. Although these mutant elastase precursors (proelastases) were produced, no appreciable processing was observed with these mutants. The possibility that autocatalysis is involved in both the processing and activation of elastase is discussed. Furthermore, by immunizing mice with vaccines made from these mutant elastase, we were able to obtain good protection against an intraperitoneal P. aeruginosa challenge.

This article has been cited by other articles:

• Bitar, A. P., Cao, M., Marquis, H. (2008). The Metalloprotease of Listeria monocytogenes Is Activated by Intramolecular Autocatalysis. J. Bacteriol. 190: 107-111 [Abstract] [Full Text]  
• Saha, S., Takeshita, F., Matsuda, T., Jounai, N., Kobiyama, K., Matsumoto, T., Sasaki, S., Yoshida, A., Xin, K.-Q., Klinman, D. M., Uematsu, S., Ishii, K. J., Akira, S., Okuda, K. (2007). Blocking of the TLR5 Activation Domain Hampers Protective Potential of Flagellin DNA Vaccine. J. Immunol. 179: 1147-1154 [Abstract] [Full Text]  
• Kooi, C., Corbett, C. R., Sokol, P. A. (2005). Functional Analysis of the Burkholderia cenocepacia ZmpA Metalloprotease. J. Bacteriol. 187: 4421-4429 [Abstract] [Full Text]  
• Ogino, H., Uchiho, T., Yokoo, J., Kobayashi, R., Ichise, R., Ishikawa, H. (2001). Role of Intermolecular Disulfide Bonds of the Organic Solvent-Stable PST-01 Protease in Its Organic Solvent Stability. Appl. Environ. Microbiol. 67: 942-947 [Abstract] [Full Text]  
• Cummins, P. M., Pabon, A., Margulies, E. H., Glucksman, M. J. (1999). Zinc Coordination and Substrate Catalysis within the Neuropeptide Processing Enzyme Endopeptidase EC 3.4.24.15. IDENTIFICATION OF ACTIVE SITE HISTIDINE AND GLUTAMATE RESIDUES. J. Biol. Chem. 274: 16003-16009 [Abstract] [Full Text]  
• Marie-Claire, C., Roques, B. P., Beaumont, A. (1998). Intramolecular Processing of Prothermolysin. J. Biol. Chem. 273: 5697-5701 [Abstract] [Full Text]  
• O'Donohue, M. J., Beaumont, A. (1996). The Roles of the Prosequence of Thermolysin in Enzyme Inhibition and Folding in Vitro. J. Biol. Chem. 271: 26477-26481 [Abstract] [Full Text]