Please use this identifier to cite or link to this item: doi:10.22028/D291-28650
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Title: Biochemical and structural characterization of CYP109A2, a vitamin D3 25-hydroxylase from Bacillus megaterium
Author(s): Abdulmughni, Ammar
Jóźwik, Ilona K.
Brill, Elisa
Hannemann, Frank
Thunnissen, Andy-Mark W. H.
Bernhardt, Rita
Language: English
Title: The FEBS journal
Volume: 284
Issue: 22
Startpage: 3881
Endpage: 3894
Publisher/Platform: Wiley-Blackwell - STM
Year of Publication: 2017
Publikation type: Journal Article
Abstract: 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 (VD3 ) metabolites are of pharmaceutical importance and are applied for the treatment of VD3 deficiency and other disorders. However, the chemical synthesis of VD3 derivatives shows low specificity and low yields. In this study, cytochrome P450 CYP109A2 from Bacillus megaterium DSM319 was expressed, purified, and shown to oxidize VD3 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 24 h and allowed to identify the main product by NMR analysis as 25-hydroxylated VD3 . 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 VD3 binding as the related VD3 hydroxylases CYP109E1 from B. 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 VD3 hydroxylation.
DOI of the first publication: 10.1111/febs.14276
URL of the first publication: https://doi.org/10.1111/febs.14276
Link to this record: hdl:20.500.11880/27680
http://dx.doi.org/10.22028/D291-28650
ISSN: 1742-464X
1742-4658
Date of registration: 6-Sep-2019
Faculty: NT - Naturwissenschaftlich- Technische Fakultät
Department: NT - Biowissenschaften
Collections:UniBib – Die Universitätsbibliographie

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