title: Characterization of cytochrome P450 CYP109E1 from Bacillus megaterium as a novel vitamin D3hydroxylase.
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), VD3was 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 VD3at 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)2VD3by CYP109E1 is derived from VD3via 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 VD3compared with the wild type activity, resulting in an approximately 2-fold increase of 25(OH)VD3production (45mgl-1day-1) compared to wild type (24.5mgl-1day-1).
authors.affiliation: Department of Biochemistry, Campus B2.2, 66123, Saarland University, Saarbrücken, Germany.
authors.forename: Ammar
authors.initials: A
authors.lastname: Abdulmughni
authors.affiliation: Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands.
authors.forename: Ilona K
authors.initials: IK
authors.lastname: Jóźwik
authors.affiliation: Department of Biochemistry, Campus B2.2, 66123, Saarland University, Saarbrücken, Germany.
authors.forename: Natalia
authors.initials: N
authors.lastname: Putkaradze
authors.affiliation: Department of Biochemistry, Campus B2.2, 66123, Saarland University, Saarbrücken, Germany.
authors.forename: Elisa
authors.initials: E
authors.lastname: Brill
authors.affiliation: Pharmaceutical Biology, Campus C2.2, 66123, Saarland University, Saarbrücken, Germany.
authors.forename: Josef
authors.initials: J
authors.lastname: Zapp
authors.affiliation: Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands.
authors.forename: Andy-Mark W H
authors.initials: AW
authors.lastname: Thunnissen
authors.affiliation: Department of Biochemistry, Campus B2.2, 66123, Saarland University, Saarbrücken, Germany. Electronic address: f.hannemann@mx.uni-saarland.de.
authors.forename: Frank
authors.initials: F
authors.lastname: Hannemann
authors.affiliation: Department of Biochemistry, Campus B2.2, 66123, Saarland University, Saarbrücken, Germany. Electronic address: ritabern@mx.uni-saarland.de.
authors.forename: Rita
authors.initials: R
authors.lastname: Bernhardt
mesh_terms: D000325:Adrenodoxin / Q000378:metabolism; D000818:Animals; D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002112:Calcifediol / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D002762:Cholecalciferol / Q000737:chemistry / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D006900:Hydroxylation; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014807:Vitamin D / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; C118756:dihydroxy-vitamin D3; D000325:Adrenodoxin; D014807:Vitamin D; D002762:Cholecalciferol; D003577:Cytochrome P-450 Enzyme System; D005287:Ferredoxin-NADP Reductase; D002112:Calcifediol
keywords: 25-Hydroxy-vitamin D(3); Bacillus megaterium; CYP109E1; Site-directed mutagenesis; Vitamin D(3); Whole-cell conversion