5OFQ

Crystal structure of substrate-free CYP109A2 from Bacillus megaterium

Summary for 5OFQ

• Entry DOI: 10.2210/pdb5ofq/pdb
• Descriptor: Cytochrome P450, PROTOPORPHYRIN IX CONTAINING FE, PENTAETHYLENE GLYCOL, ... (5 entities in total)
• Functional Keywords: bacterial proteins, bacillus megaterium, bacillus, cytochrome p-450 enzyme system, cytochrome p450, hydroxylation, heme, oxidation-reduction, 25-hydroxyvitamin d3, biocatalysis, calcifediol, cholecalciferol, oxidoreductase
• Biological source: Bacillus megaterium (strain DSM 319)
• Total number of polymer chains: 4
• Total formula weight: 191891.11

Authors

Jozwik, I.K.,Thunnissen, A.M.W.H. (deposition date: 2017-07-11, release date: 2017-09-27, Last modification date: 2024-01-17)

Primary citation

Abdulmughni, A.,Jozwik, I.K.,Brill, E.,Hannemann, F.,Thunnissen, A.W.H.,Bernhardt, R.
Biochemical and structural characterization of CYP109A2, a vitamin D3 25-hydroxylase from Bacillus megaterium.
FEBS J., 284:3881-3894, 2017

PubMed 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 D (VD ) metabolites are of pharmaceutical importance and are applied for the treatment of VD deficiency and other disorders. However, the chemical synthesis of VD derivatives shows low specificity and low yields. In this study, cytochrome P450 CYP109A2 from Bacillus megaterium DSM319 was expressed, purified, and shown to oxidize VD 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 VD . Product yield amounted to 54.9 mg·L ·day , 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 VD binding as the related VD 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 VD hydroxylation.

PubMed: 28940959
DOI: 10.1111/febs.14276

Experimental method

X-RAY DIFFRACTION (2.7 Å)