Identification of the catalytic triad of the protein D2 protease in Pseudomonas aeruginosa.
We reported recently that protein D2 (OprD) porin of Pseudomonas aeruginosa bears protease activity (FEBS Letters 394, 179-182, 1996). To identify the catalytic residues of OprD, we introduced the site-directed mutations replacing the putative catalytic triad His156, Asp208, and Ser296 with glutamine, asparagine, and alanine, respectively. The OprD proteins purified from the chromosomal oprD-deficient mutants harboring the plasmids encoding the site-directed mutations showed protease activity less than 0.1% of that of the wild-type OprD. These site-directed mutageneses caused undetectable changes in the pore-forming activity of OprD as measured by single-channel conductance by the planar lipid bilayer. The minimum inhibitory concentration of imipenem in mutants having the replaced catalytic triads was identical with that in the wild-type strain. On the other hand, introduction of the mutation at His367 replacing with glutamine, the site that is supposed to be unrelated to the catalytic sites, showed the unchanged protease activity. These results unequivocally demonstrate that OprD is the protease bearing porin and catalyzes the reaction at His156, Asp208, and Ser296 residues.
Yoshihara
E
E
Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Japan.
Yoneyama
H
H
Ono
T
T
Nakae
T
T
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D015780:Carbapenems / Q000494:pharmacology; D002384:Catalysis; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine / Q000235:genetics; D006639:Histidine / Q000235:genetics; D015378:Imipenem / Q000494:pharmacology; D007473:Ion Channels / Q000187:drug effects / Q000235:genetics; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed; D018272:Porins / Q000235:genetics / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D012694:Serine / Q000235:genetics
D015780:Carbapenems; C094221:DX 8739; D007473:Ion Channels; D018272:Porins; D005973:Glutamine; C077157:OprD protein, Pseudomonas aeruginosa; D001224:Aspartic Acid; D012694:Serine; D006639:Histidine; D001216:Asparagine; D015378:Imipenem; D010450:Endopeptidases; D000409:Alanine