Construction and engineering of a thermostable self-sufficient cytochrome P450.
CYP175A1 is a thermophilic cytochrome P450 and hydroxylates beta-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): H(2)N-CYP175A1-Fdx-FNR-COOH (175FR) and H(2)N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V(max) value for beta-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k(m) values of these enzymes were similar. 175RF retained 50% residual activity even at 80 degrees 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.
Mandai
Takao
T
Department of Bioscience, Nanobiotechnology Research Center, School of Science and Technology, Kwansei Gakuin University, Sanda, Japan.
Fujiwara
Shinsuke
S
Imaoka
Susumu
S
D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D015202:Protein Engineering; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System