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Biochemical and structural characterization of CYP 109A2, a vitamin D 3 25‐hydroxylase from Bacillus megaterium
Author(s) -
Abdulmughni Ammar,
Jóźwik Ilona K.,
Brill Elisa,
Hannemann Frank,
Thunnissen AndyMark W. H.,
Bernhardt Rita
Publication year - 2017
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.14276
Subject(s) - bacillus megaterium , cytochrome p450 , chemistry , stereochemistry , enzyme kinetics , reductase , metabolite , biochemistry , enzyme , stereoselectivity , selectivity , active site , catalysis , biology , genetics , bacteria
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 D 3 (VD 3 ) metabolites are of pharmaceutical importance and are applied for the treatment of VD 3 deficiency and other disorders. However, the chemical synthesis of VD 3 derivatives shows low specificity and low yields. In this study, cytochrome P450 CYP 109A2 from Bacillus megaterium DSM 319 was expressed, purified, and shown to oxidize VD 3 with high regio‐selectivity. The in vitro conversion, using cytochrome P450 reductase (Bm CPR ) and ferredoxin (Fdx2) from the same strain, showed typical Michaelis–Menten reaction kinetics. A whole‐cell system in B. megaterium overexpressing CYP 109A2 reached 76 ± 5% conversion after 24 h and allowed to identify the main product by NMR analysis as 25‐hydroxylated VD 3 . 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 CYP 109A2 was determined at 2.7 Å resolution, displaying an open conformation. Structural analysis predicts that CYP 109A2 uses a highly similar set of residues for VD 3 binding as the related VD 3 hydroxylases CYP 109E1 from B. megaterium and CYP 107 BR 1 (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 VD 3 hydroxylation. Database The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession code 5OFQ (substrate‐free CYP 109A2). Enzymes Cytochrome P450 monooxygenase CYP109A2, EC 1.14.14.1 , UniProt ID : D5DF88 , Ferredoxin, UniProt ID : D5DFQ0 , cytochrome P450 reductase, EC 1.8.1.2 , UniProt ID : D5DGX1 .