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.
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.
Kawamoto
S
S
Department of Bacteriology, Yokohama City University School of Medicine, Japan.
Shibano
Y
Y
Fukushima
J
J
Ishii
N
N
Morihara
K
K
Okuda
K
K
D000818:Animals; D001428:Bacterial Vaccines / Q000276:immunology; D001483:Base Sequence; D001665:Binding Sites; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010196:Pancreatic Elastase / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011552:Pseudomonas Infections / Q000201:enzymology*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013820:Thermolysin / Q000737:chemistry; D014614:Vaccines, Synthetic / Q000276:immunology
D001428:Bacterial Vaccines; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; D010196:Pancreatic Elastase; D013820:Thermolysin