Regio‐ and stereoselective steroid hydroxylation by CYP109A2 from Bacillus megaterium explored by X‐ray crystallography and computational modeling

Abstract

The P450 monooxygenase CYP109A2 from Bacillus megaterium DSM319 was previously found to convert vitamin D3 (VD3) to 25‐hydroxyvitamin D3. Here, we show that this enzyme is also able to convert testosterone in a highly regio‐ and stereoselective manner to 16β‐hydroxytestosterone. To reveal the structural determinants governing the regio‐ and stereoselective steroid hydroxylation reactions catalyzed by CYP109A2, two crystal structures of CYP109A2 were solved in similar closed conformations, one revealing a bound testosterone in the active site pocket, albeit at a nonproductive site away from the heme‐iron. To examine whether the closed crystal structures nevertheless correspond to a reactive conformation of CYP109A2, docking and molecular dynamics (MD) simulations were performed with testosterone and vitamin D3 (VD3) present in the active site. These MD simulations were analyzed for catalytically productive conformations, the relative occurrences of which were in agreement with the experimentally determined stereoselectivities if the predicted stability of each carbon‐hydrogen bond was taken into account. Overall, the first‐time determination and analysis of the catalytically relevant 3D conformation of CYP109A2 will allow for future small molecule ligand screening in silico, as well as enabling site‐directed mutagenesis toward improved enzymatic properties of this enzyme.

Recent developments in the enzymatic modifications of steroid scaffolds

“…61 The novel CYP17A2 from Mastacembelus armatus (MA_CYP17A2), identified by genome mining, efficiently performed the C-17α hydroxylation of PG (12) (120.9 ± 4.2 mg L −1 in 7 days). 62 CYP109A2 from Bacillus megaterium DSM319 catalyzed the 16β-hydroxylation of TES (10), 63 while the human P450 11B2 catalyzed the C-11β hydroxylation of 11-deoxycorticosterone (28) and PG (12). 64 2.2 αKG OXs α-Ketoglutarate-dependent oxygenases (αKG OXs) are metalloenzymes with a non-heme Fe 2+ ion in the active center, and are widely distributed in both primary and secondary metabolic pathways.…”