Putative functions of phenylalanine-350 of Pseudomonas putida cytochrome P-450cam |
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| Affiliation: | 1. Department of Microbiology, Faculty of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka 812, Japan;2. Department of Food and Nutrition, Nakamura Gakuen College, Jonan-ku, Fukuoka 814-01, Japan;3. Department of Bioengineering, Faculty of Engineering, Soka University, Tangi-cho 1–236, Hachioji, Tokyo 192, Japan;1. State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, Xining, 810016, China;2. School of Chemical Engineering, Qinghai University, Xining, 810016, China;1. School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, PR China;2. Department of Chemistry, Institute for Advanced Study, Division of Biomedical Engineering, Division of Life Science, State Key Laboratory of Molecular Neuroscience, Institute of Molecular Functional Materials, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China;3. State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fu Zhou, Fujian 350002, PR China;1. School of Chemical Engineering and Technology, Tianjin University, Tianjin, PR China;2. Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), PR China;3. Tianjin Engineering Research Center of Functional Fine Chemicals, Tianjin, PR China;1. Magneto-plasmonic Lab, Laser and Plasma Research Institute, Shahid Beheshti University, Tehran, Iran;2. Department of Laser Physics, College of Science for Woman, University of Babylon, Babylon, Iraq;3. Department of Physics, College of Science, University of Baghdad, Baghdad, Iraq |
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| Abstract: | 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 reconsituted 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. |
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