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

Abdulmughni, A., Jóźwik, I. K., Brill, E., Hannemann, F., Thunnissen, A-M. W. H. & Bernhardt, R. 22-Sep-2017 In : Febs Journal.

Research output: Scientific - peer-reviewArticle

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DOI

Cytochrome P450 enzymes are increasingly investigated due to their potential application as biocatalysts with high regio- and/or stereo-selectivity and under mild conditions. Vitamin D3 metabolites are of pharmaceutical importance and are applied for the treatment of vitamin D3 deficiency and other disorders. However, the chemical synthesis of vitamin D3 derivatives shows low specificity and low yields. In this study, cytochrome P450 CYP109A2 from Bacillus megaterium DSM319 was expressed, purified and shown to oxidize vitamin D3 with high regio-selectivity. The in vitro conversion, using cytochrome P450 reductase (BmCPR) and ferredoxin (Fdx2) from the same strain, showed typical Michaelis-Menten reaction kinetics. A whole-cell system in B. megaterium overexpressing CYP109A2 reached 76 ± 5% conversion after 24h and allowed to identify the main product by NMR analysis as 25-hydroxylated vitamin D3 . Product yield amounted to 54.9 mg L(-1) day(-1) , rendering the established whole-cell system as a highly promising biocatalytic route for the production of this valuable metabolite. The crystal structure of substrate-free CYP109A2 was determined at 2.7 Å resolution, displaying an open conformation. Structural analysis predicts that CYP109A2 uses a highly similar set of residues for vitamin D3 binding as the related vitamin D3 hydroxylases CYP109E1 from Bacillus megaterium and CYP107BR1 (Vdh) from Pseudonocardia autotrophica. However, the folds and sequences of the BC loops in these three P450s are highly divergent, leading to differences in the shape and apolar/polar surface distribution of their active site pockets, which may account for the observed differences in substrate specificity and the regio-selectivity of vitamin D3 hydroxylation. This article is protected by copyright. All rights reserved.

Original languageEnglish
JournalFebs Journal
StateAccepted/In press - 22-Sep-2017

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