Highly regio- and stereoselective hydroxylation of vitamin D2 by CYP109E1

Vitamin D2 is a form of vitamin D derived from mushrooms and plants which is structurally modified in the body due to the action of several enzymes. The resulting metabolites represent important compounds with potential bioactive properties. However, they are poorly studied and their availability is mostly limited. In order to identify new enzymes capable of producing vitamin D2 metabolites, we investigated a bacterial P450 monooxygenase, CYP109E1, which was previously shown to be a vitamin D3 hydroxylase. It was found that CYP109E1 catalyzes a vitamin D2 two-step hydroxylation at positions C24 and C25 resulting in the generation of 24(R),25-diOH VD2. Interestingly, the enzyme showed high selectivity towards vitamin D2, whereas it showed an unselective product pattern for the structurally similar vitamin D3. Our docking results for vitamin D2 and D3 revealed favorable hydroxylation positions for both substrates and suggested an explanation for the high selectivity of CYP109E1 towards vitamin D2. In addition, we established a whole-cell biocatalyst expressing CYP109E1 in Bacillus megaterium to produce 24(R),25-diOH VD2 and a production yield of 12.3 ± 1.2 mg/L was obtained after 48 h. To the best of our knowledge, this is the first report on the generation of 24(R),25-diOH VD2 by a microbial biocatalyst allowing a low-cost and eco-friendly production of this pharmaceutically interesting and expensive metabolite from the relatively cheap substrate, VD2. •Identification of CYP109E1 from Bacillus megaterium as a highly selective vitamin D2 (VD2) hydroxylase.•CYP109E1 carries out two hydroxylations and forms the physiological VD metabolite 24(R),25-diOH VD2 as a final product.•Docking studies using the crystal structure explained the high selectivity of CYP109E1 towards VD2.•First microbial P450 system for the production of 24(R),25-diOH VD2 was established.•Production of 12.3 ± 1.2 mg/L 24(R),25-diOH VD2 by a CYP109E1-expressing whole-cell system was accomplished.