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doi:10.22028/D291-28652
Titel: | Characterization of cytochrome P450 CYP109E1 from Bacillus megaterium as a novel vitamin D3 hydroxylase |
VerfasserIn: | Abdulmughni, Ammar Jóźwik, Ilona K. Putkaradze, Natalia Brill, Elisa Zapp, Josef Thunnissen, Andy-Mark W. H. Hannemann, Frank Bernhardt, Rita |
Sprache: | Englisch |
Titel: | Journal of Biotechnology |
Bandnummer: | 243 |
Startseite: | 38 |
Endseite: | 47 |
Verlag/Plattform: | Elsevier |
Erscheinungsjahr: | 2017 |
Dokumenttyp: | Journalartikel / Zeitschriftenartikel |
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). |
DOI der Erstveröffentlichung: | 10.1016/j.jbiotec.2016.12.023 |
URL der Erstveröffentlichung: | https://doi.org/10.1016/j.jbiotec.2016.12.023 |
Link zu diesem Datensatz: | hdl:20.500.11880/27695 http://dx.doi.org/10.22028/D291-28652 |
ISSN: | 1873-4863 0168-1656 |
Datum des Eintrags: | 6-Sep-2019 |
Fakultät: | NT - Naturwissenschaftlich- Technische Fakultät |
Fachrichtung: | NT - Biowissenschaften |
Professur: | NT - Prof. Dr. Bruce Morgan |
Sammlung: | SciDok - Der Wissenschaftsserver der Universität des Saarlandes |
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