Engineering of P450pyr hydroxylase for the highly regio- and enantioselective subterminal hydroxylation of alkanes

Abstract

Terminal-selective cytochrome P450pyr has been successfully engineered through directed evolution for the subterminal hydroxylation of alkanes with excellent regio- and enantioselectivity. A sensitive colorimetric high-throughput screening (HTS) assay was developed for the measurement of both the regioselectivity and the enantioselectivity of a hydroxylation reaction. By using the HTS assay and iterative saturation mutagenesis, sextuple-mutant P450pyrSM1 was created for the hydroxylation of n-octane (1) to give (S)-2-octanol (2) with 98 % ee and >99 % subterminal selectivity. The engineered P450 is the first enzyme for this type of highly selective alkane hydroxylation, being useful for the Ci-H activation and functionalization of alkanes and the preparation of enantiopure alcohols. Molecular modeling provided structure-based understanding of the fully altered regioselectivity and the excellent enantioselectivity. Another sextuple-mutant P450pyrSM2 catalyzed the hydroxylation of propylbenzene (3) to afford (S)-1-phenyl-2-propanol (4) with 95 % ee and 98 % subterminal selectivity. Get a handle on it: Highly regio- and enantioselective subterminal hydroxylation of n-octane and propylbenzene was observed with P450 enzymes obtained by the directed evolution of terminal-selective P450pyr hydroxylase (see scheme). The engineered enzymes with their fully altered selectivities are useful for the functionalization of alkanes and the preparation of enantiomerically pure alcohols. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.