Production of an active form of vitamin D by genetically engineered CYP105A1

Our previous studies revealed that CYP105A1 can convert vitamin D (VD3) to its active form， 1α，25-dihydroxyvitamin D (1，25D3). Site-directed mutagenesis of CYP105A1 based on its crystal structure dramatically enhanced its activity; the activity of double variants R73A/R84A and R73A/R84V was more than 100-fold higher than that of the wild type of CYP105A1. In contrast， these variants had a low ability to convert vitamin D (VD2) to 1α，25-dihydroxyvitamin D (1，25D2)， whereas they catalyzed the sequential hydroxylation at positions C25 and C26 to produce 25，26D2. A comparison of the docking models of 25D2 and 25D3 into the substrate-binding pocket of R73A/R84A suggests that the side chain of the Met239 inhibits the binding of 25D2 for 1α-hydroxylation. Therefore， the Met239 residue of R73A/R84A was substituted for Ala. As expected， the triple variant R73A/R84A/M239A showed a 22-fold higher 1α-hydroxylation activity towards 25D2. To the best of our knowledge， this is the first report on the generation of microbial cytochrome P450 that converts VD2 to 1，25D2 via 25D2.