title: Directed evolution of cytochrome P450 for sterol epoxidation.
abstract: 16,17-Epoxysterol plays an important role in pharmaceutical steroid synthesis. To investigate the potential application of cytochrome P450 for epoxysterol synthesis, an approach to the epoxidation of 16,17-epoxysterol, based on directed evolution of cytochrome P450 BM-3, was developed. This comprised random gene mutagenesis for optimizing the activity of P450 BM-3 for epoxidation of hydrophobic sterol, followed by the 7-ethoxycoumarin de-ethylation assay for general enzyme activity detection and the modified picric acid assay for epoxidation activity screening. By the two-step screening, one mutant from 792 clones showed specific substrate activity of converting progesterone to 16,17-epoxysterol, which validated the possibility to evolve the cytochrome P450 for the synthesis of steroidal epoxides.
authors.affiliation: School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Jiang
authors.forename: Ran
authors.initials: R
authors.lastname: Tu
authors.forename: Peng
authors.initials: P
authors.lastname: Bai
authors.forename: Qinhong
authors.initials: Q
authors.lastname: Wang
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001711:Biotransformation; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000378:metabolism*; D011374:Progesterone / Q000378:metabolism; D013261:Sterols / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D013261:Sterols; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases