Regio-and Enantioselective Oxidation by CYP108N12: Substrate Scope and Specificity

Abstract

Growing demand for efficient, safe and economically viable catalytic processes has driven increasing interest in using enzymes for large-scale, biocatalytic transformations. Cytochromes P450 are enantio-, regio-and chemo-selective enzymes renowned for catalysing oxygen insertion into unactivated C-H bonds across a wide range of compounds including steroids, alkanes and terpenes. Despite their impressive catalytic ability and diverse substrate scope, these enzymes are somewhat limited in utility by their requirement for auxiliary redox partner proteins as well as stoichiometric amounts of an expensive nicotinamide cofactor for in vitro catalytic turnovers. CYP108N12 from the bacterium Rhodococcus globerulus has previously been found to initiate p-cymene biodegradation through benzylic methyl hydroxylation.Subsequent investigation revealed that the efficiency of in vitro catalysis was greatly increased when supported by its native ferredoxin redox partner, cymredoxin, resulting in high yields of a monohydroxylated product. In this work, a bicistronic construct of CYP108N12 and cymredoxin was created and utilised to perform larger scale, in vivo turnovers in Escherichia coli. This system was found to regio-, chemo-and enantioselectively oxidise monoterpenes as well as a range of 1,4-disubstituted benzenes. The latter transformations included the enantioselective monohydroxylation of para-substituted ethyl and propyl benzenes. Additionally, CYP108N12 demonstrated the ability to hydroxylate 1-bromo-4-isopropylbenzene at the isopropyl methine and methyl positions, resulting in the formation of (R)-2-(4bromophenyl)propane-1,2-diol as well as the two singly hydroxylated products. With its ease of production, diverse substrate range, high activity and enantioselectivity the recombinant CYP108N12 system is an attractive, selective monooxygenase with high potential for biotechnological utility, especially in the chemical catalysis space.