Controlling the Regioselectivity of Fatty Acid Hydroxylation (C-10) at alpha- and beta-Position by CYP152A1 (P450Bs beta) Variants

摘要

Regioselective hydroxylation on inactivated C-H bonds is among the dream reactions of organic chemists. Cytochrome P450 enzymes (CYPs) perform this reaction in general with high regio- and stereoselectivity (e. g. for steroids as substrates). Furthermore, enzyme engineering may allow to tune the properties of the enzyme. Regioselective hydroxylation of shorter or linear molecules (fatty acids), however, remains challenging even with this enzyme class, due to the high similarity of the substrate's backbone carbons and their conformational flexibility. CYPs hydroxylating fatty acids selectively in the chemically more distinct alpha- or omega- position are well described. In contrast, selective in-chain hydroxylation of fatty acids lacks precedence. The peroxygenase CYP152A1 (P450Bs beta) is a family member that displays fatty acid hydroxylation at both, the alpha- and beta-position, with preference for the alpha-position. Herein we report the influence of hydrophobic active site residues on the hydroxylation pattern of this enzyme. By site directed mutagenesis and combination of the libraries, double and triple mutation variants were identified, which hydroxylated decanoic acid (C-10) with improved regio-selectivity in the beta-position. Variants were identified with a 10-fold increase of the beta-regioselectivity (expressed as alpha/beta-ratio) compared to the wild type. In total 103 variants of CYP152A1 (P450Bs beta) were investigated.