Characterization of cytochrome P450 CYP109E1 from Bacillus megaterium as a novel vitamin D3 hydroxylase

Abstract

In this study the ability of CYP109E1 from Bacillus megaterium to metabolize vitamin D₃ (VD₃) was investigated. In an in vitro system using bovine adrenodoxin reductase (AdR) and adrenodoxin (Adx_4-108), VD₃ 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 VD₃ at carbons C-24 and C-25, resulting in the formation of 24(S)-hydroxyvitamin D₃ (24S(OH)VD₃), 25-hydroxyvitamin D₃ (25(OH)VD₃) and 24S,25-dihydroxyvitamin D₃ (24S,25(OH)₂VD₃). Through time dependent whole-cell conversion of VD₃, we identified that the formation of 24S,25(OH)₂VD₃ by CYP109E1 is derived from VD₃ via the intermediate 24S(OH)VD₃. 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 VD₃ compared with the wild type activity, resulting in an approximately 2-fold increase of 25(OH)VD₃ production (45mgl^-1day^-1) compared to wild type (24.5mgl^-1day^-1).

Keywords: 25-Hydroxy-vitamin D(3); Bacillus megaterium; CYP109E1; Site-directed mutagenesis; Vitamin D(3); Whole-cell conversion.