Characterization of cytochrome P450 CYP109E1 from Bacillus megaterium as a novel vitamin D hydroxylase.

In this study the ability of CYP109E1 from Bacillus megaterium to metabolize vitamin D (VD) was investigated. In an in vitro system using bovine adrenodoxin reductase (AdR) and adrenodoxin (Adx), VD was converted by CYP109E1 into several products. Furthermore, a whole-cell system in B. megaterium MS941 was established. The new system showed a conversion of 95% after 24h. By NMR analysis it was found that CYP109E1 catalyzes hydroxylation of VD at carbons C-24 and C-25, resulting in the formation of 24(S)-hydroxyvitamin D (24S(OH)VD), 25-hydroxyvitamin D (25(OH)VD) and 24S,25-dihydroxyvitamin D (24S,25(OH)VD). Through time dependent whole-cell conversion of VD, we identified that the formation of 24S,25(OH)VD by CYP109E1 is derived from VD via the intermediate 24S(OH)VD. Moreover, using docking analysis and site-directed mutagenesis, we identified important active site residues capable of determining substrate specificity and regio-selectivity. HPLC analysis of the whole-cell conversion with the I85A-mutant revealed an increased selectivity towards 25-hydroxylation of VD compared with the wild type activity, resulting in an approximately 2-fold increase of 25(OH)VD production (45mglday) compared to wild type (24.5mglday).