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 D 3 (VD 3 ), the precursor of vitamin D (1α,25‐dihydroxyVD 3 ). 25‐HydroxyVD 3 , the circulating form of vitamin D, is a more effective supplement than VD 3 but its synthesis is complex. We report here the engineering of cytochrome P450 BM3 (CYP102A1) for the selective oxidation of VD 3 to 25‐hydroxyVD 3 . Long‐range effects of the substrate‐channel mutation Glu435Ile promoted binding of the VD 3 side chain close to the heme, enhancing VD 3 oxidation activity that reached 6.62 g of 25‐hydroxyVD 3 isolated from a 1‐litre scale reaction (69.1 % yield; space‐time‐yield 331 mg/L/h).