Laboratory evolution of cytochrome P450 BM-3 monooxygenase for organic cosolvents

Abstract

Cytochrome P450 BM‐3 (EC 1.14.14.1) catalyzes the hydroxylation and/or epoxidation of a broad range of substrates, including alkanes, alkenes, alcohols, fatty acids, amides, polyaromatic hydrocarbons, and heterocycles. For many of these notoriously water‐insoluble compounds, P450 BM‐3's K_m values are in the millimolar range. Polar organic cosolvents are therefore added to increase substrate solubility and achieve high catalytic efficiency. Using P450 BM‐3 as a catalyst for these important transformations requires that we improve its ability to tolerate the cosolvents. By directed evolution, we improved the activity of P450 BM‐3 in the presence of dimethylsulfoxide (DMSO) and tetrahydrofuran (THF), achieving increases in specific activity up to 10‐fold in 2% (v/v) THF and 6‐fold in 25% (v/v) DMSO. The engineered P450 BM‐3's are also significantly more resistant to acetone, acetonitrile, dimethylformamide, and ethanol as cosolvents in the reaction.