Construction and engineering of a thermostable self-sufficient cytochrome P450 |
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Authors: | Takao Mandai |
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Institution: | Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337, Japan |
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Abstract: | CYP175A1 is a thermophilic cytochrome P450 and hydroxylates β-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP+ reductase (FNR): H2N-CYP175A1-Fdx-FNR-COOH (175FR) and H2N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The Vmax value for β-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the km values of these enzymes were similar. 175RF retained 50% residual activity even at 80 °C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate. |
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Keywords: | Ferredoxin-NADP+ reductase Ferredoxin CYP175A1 β-Carotene hydroxylation Fusion protein Testosterone hydroxylation Protein engineering |
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