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

• Published in: Chemistry : a European journal Vol. 30; no. 51; pp. e202401487 - n/a
• Main Authors: Chen, Wenyu, Lynch, Jamie N. C., Bustamante, Claudia, Zhang, Yuan, Wong, Luet L.
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley Subscription Services, Inc 11.09.2024
• Subjects: Calciferol; Cytochrome; Cytochromes; C−H activation; Dietary supplements; Monooxygenases; Mutation; Oxidation; P450 enzymes; Protein engineering; Substrates; Vitamin D; Vitamin D3; Vitamin D3 oxidation; Vitamin deficiency
• ISSN: 0947-6539; 1521-3765; 1521-3765
• DOI: 10.1002/chem.202401487

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 P450BM3 (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). Long‐range effects from mutation of a substrate access tunnel residue selected by docking‐guided design of cytochrome P450BM3 (CYP102A1) to loosen the interaction between two secondary structure elements reshaped the substrate binding pocket and increased the activity for the oxidation vitamin D3 to 25‐hydroxy‐vitamin D3, an important human and animal health supplement.