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J Biotechnol. 2017 Feb 10;243:38-47. doi: 10.1016/j.jbiotec.2016.12.023. Epub 2016 Dec 30.

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

Author information

1
Department of Biochemistry, Campus B2.2, 66123, Saarland University, Saarbrücken, Germany.
2
Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands.
3
Pharmaceutical Biology, Campus C2.2, 66123, Saarland University, Saarbrücken, Germany.
4
Department of Biochemistry, Campus B2.2, 66123, Saarland University, Saarbrücken, Germany. Electronic address: f.hannemann@mx.uni-saarland.de.
5
Department of Biochemistry, Campus B2.2, 66123, Saarland University, Saarbrücken, Germany. Electronic address: ritabern@mx.uni-saarland.de.

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 (Adx4-108), 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

PMID:
28043840
DOI:
10.1016/j.jbiotec.2016.12.023
[Indexed for MEDLINE]

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