Selective Oxidation of Vitamin D3 Enhanced by Long-Range Effects of a Substrate Channel Mutation in Cytochrome P450BM3 (CYP102A1)

Abstract

Vitamin D deficiency affects nearly half the population, with many requiring or opting for supplements with vitamin D₃ (VD₃), the precursor of vitamin D (1α,25-dihydroxyVD₃). 25-HydroxyVD₃, the circulating form of vitamin D, is a more effective supplement than VD₃ but its synthesis is complex. We report here the engineering of cytochrome P450_BM3 (CYP102A1) for the selective oxidation of VD₃ to 25-hydroxyVD₃. Long-range effects of the substrate-channel mutation Glu435Ile promoted binding of the VD₃ side chain close to the heme, enhancing VD₃ oxidation activity that reached 6.62 g of 25-hydroxyVD₃ isolated from a 1-litre scale reaction (69.1 % yield; space-time-yield 331 mg/L/h).

Keywords: C−H activation; Monooxygenases; P450 enzymes; Protein engineering; Vitamin D3 oxidation.