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

Biochemical and structural characterization of CYP109A2, a vitamin D3 25‐hydroxylase from Bacillus megaterium

“…These results demonstrate that the CYP109A2 in vitro activity toward VD 3 is lower than that of other previously characterized bacterial VD 3 hydroxylases . In addition, CYP109A2 displays a lower substrate conversion ratio in comparison to its most closely related homolog CYP109E1, which was shown to convert ~ 90% of VD 3 under the same conditions (when supported by the AdR–Adx 4–108 pair) . The affinity for VD 3 is also lower in CYP109A2 as compared with wild‐type CYP105A1 or Vdh, which showed K M values of 0.54 ± 0.09 μ m and 13.5 ± 3.7 μ m , respectively .…”

“…Thus far, only a few bacterial P450 enzymes have been reported that hydroxylate VD 3 to produce the desired 25(OH)VD 3 metabolite, like CYP105A1 from Streptomyces griseolus [11] or CYP107BR1 (Vdh) from Pseudonocardia autotrophica [12]. Most recently, our investigations led to the identification and characterization of another bacterial VD 3hydroxylase, cytochrome P450 CYP109E1 from Bacillus megaterium DSM319 [13]. The improvement of activity and/or regio-selectivity of the three abovementioned enzymes by protein engineering was supported by the knowledge of their structure-function relationships, due to determination of their crystal structures in both the open, substrate-free and closed, substrate-bound states [14][15][16][17][18].…”

“…Similar to CYP109E1, CYP109A2 exhibits 25-hydroxylation activity toward VD 3 , however, with significantly higher regio-selectivity. A whole-cell B. megaterium-based VD 3 oxidation system was established for CYP109A2 and resulted in higher yields of 25(OH)VD 3 as compared with the previously reported most productive variant of CYP109E1, i.e., mutant I85A [13]. The three-dimensional structure of CYP109A2 was obtained by X-ray crystallography and compared with the structures of other bacterial P450s that convert VD 3 , suggesting putative structural features associated with differences in substrate specificity and regio-selectivity.…”

CYP109E1 from Bacillus megaterium Acts as a 24‐ and 25‐Hydroxylase for Cholesterol

“…CYP109E1 is a P450 from Bacillus megaterium strain DSM319 capable of converting chemically different compounds, including testosterone, which is a steroid hormone derived from cholesterol . Furthermore, CYP109E1 is able to hydroxylate cholecalciferol (vitamin D 3 ), another derivative of cholesterol, at side‐chain positions C24 and C25 . As a consequence, the ability of CYP109E1 to use cholesterol as a substrate was investigated in this study.…”

“…To improve the selectivity of this enzyme towards one of these oxysterols, the active site could be engineered through sitedirected mutagenesis supported by docking studies because the crystal structure of CYP109E1 was previously solved . Such approaches were previously demonstrated to be an effective strategy for the improvement of product specificity of P450s …”