Putative functions of phenylalanine-350 of Pseudomonas putida cytochrome P-450cam.
Cytochrome P-450cam hydroxylates d-camphor, using molecular oxygen and reducing equivalents transferred via putidaredoxin. We constructed mutant genes in which Phe-350 of P-450cam was replaced by Leu, Tyr, or His by site-directed mutagenesis, expressed them in Escherichia coli, purified the mutant proteins, and compared their enzymic properties with those of the wild type P-450cam. NADH oxidation rate of the Tyr mutant in the reconstituted system with putidaredoxin and putidaredoxin reductase was similar to that of the wild type enzyme, while the Leu mutant and the His mutant showed 67% and 17% activity of that of the wild type, respectively. The affinities of these mutant proteins for camphor and the oxidized form of putidaredoxin were much the same as those of the wild type protein. Rate constants for the reduction reaction of P-450cam by reduced putidaredoxin, a physiological electron donor for P-450cam, of Tyr and His mutants were much the same as that of the wild type enzyme, whereas the Leu mutant showed approx. half that of the wild type. Thus, the aromatic ring of Phe-350 of P-450cam probably contributes to enhancing efficiency of the electron transfer yet does not seem to be essential for the reaction.
Yasukochi
T
T
Department of Microbiology, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Okada
O
O
Hara
T
T
Sagara
Y
Y
Sekimizu
K
K
Horiuchi
T
T
D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry*; D016958:Pseudomonas putida / Q000201:enzymology*
D005288:Ferredoxins; D006418:Heme; D010649:Phenylalanine; C100137:putidaredoxin; D003577:Cytochrome P-450 Enzyme System