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Putative functions of phenylalanine-350 of Pseudomonas putida cytochrome P-450cam
1Department of Microbiology, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
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 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.
Insights
Phenylalanine-350 in cytochrome P-450cam enhances electron transfer efficiency but is not essential for camphor hydroxylation. Mutants show varied activity, with Tyr and His variants retaining significant function.
Area of Science:
- Biochemistry
- Enzymology
- Protein Engineering
Background:
- Cytochrome P-450cam catalyzes d-camphor hydroxylation using molecular oxygen.
- Electron transfer is mediated by putidaredoxin and putidaredoxin reductase.
Purpose of the Study:
- To investigate the role of Phenylalanine-350 (Phe-350) in the catalytic efficiency of Cytochrome P-450cam.
- To understand the contribution of the aromatic ring of Phe-350 to electron transfer and substrate binding.
Main Methods:
- Site-directed mutagenesis was used to create P-450cam mutants with Phe-350 replaced by Leucine (Leu), Tyrosine (Tyr), or Histidine (His).
- Mutant proteins were expressed in Escherichia coli and purified.
- Enzymic properties, including NADH oxidation rates, substrate affinity, and electron transfer kinetics, were compared to the wild-type enzyme.
Main Results:
- The Tyr mutant exhibited similar NADH oxidation rates and electron transfer kinetics to the wild-type enzyme.
- The Leu and His mutants showed reduced activity, with Leu mutant having 67% and His mutant 17% of wild-type activity.
- Mutant proteins displayed comparable affinities for d-camphor and oxidized putidaredoxin as the wild-type enzyme.
Conclusions:
- The aromatic ring of Phe-350 in P-450cam likely enhances electron transfer efficiency.
- Phe-350 is not strictly essential for the catalytic activity of P-450cam, as demonstrated by the functional Tyr and His mutants.