Structural basis of steroid binding and oxidation by the cytochrome P450 CYP109E1 from Bacillus megaterium

Abstract

Cytochrome P450 monooxygenases (P450s) are attractive enzymes for the pharmaceutical industry, in particular, for applications in steroidal drug synthesis. Here, we report a comprehensive functional and structural characterization of CYP109E1, a novel steroid‐converting cytochrome P450 enzyme identified from the genome of Bacillus megaterium DSM319. In vitro and whole‐cell in vivo turnover experiments, combined with binding assays, revealed that CYP109E1 is able to hydroxylate testosterone at position 16β. Related steroids with bulky substituents at carbon C17, like corticosterone, bind to the enzyme without being converted. High‐resolution X‐ray structures were solved of a steroid‐free form of CYP109E1 and of complexes with testosterone and corticosterone. The structural analysis revealed a highly dynamic active site at the distal side of the heme, which is wide open in the absence of steroids, can bind four ordered corticosterone molecules simultaneously, and undergoes substantial narrowing upon binding of single steroid molecules. In the crystal structures, the single bound steroids adopt unproductive binding modes coordinating the heme‐iron with their C3‐keto oxygen. Molecular dynamics (MD) simulations suggest that the steroids may also bind in ~180° reversed orientations with the C16 carbon and C17‐substituents pointing toward the heme, leading to productive binding of testosterone explaining the observed regio‐ and stereoselectivity. The X‐ray structures and MD simulations further identify several residues with important roles in steroid binding and conversion, which could be confirmed by site‐directed mutagenesis. Taken together, our results provide unique insights into the CYP109E1 activity, substrate specificity, and regio/stereoselectivity.DatabaseThe atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 5L90 (steroid‐free CYP109E1), 5L91 (CYP109E1‐COR4), 5L94 (CYP109E1‐TES), and 5L92 (CYP109E1‐COR).EnzymesCytochrome P450 monooxygenase CYP109E1, EC 1.14.14.1, UniProt ID: D5DKI8, Adrenodoxin reductase EC 1.18.1.6.

Biochemical and structural characterization of CYP109A2, a vitamin D3 25‐hydroxylase from Bacillus megaterium

“…Bioinformatic mining of the B. megaterium DSM319 genome previously led to identification of four genes putatively encoding for cytochrome P450 enzymes (CYP102A1, CYP106A1, CYP109E1, and CYP109A2) . Here, we focus on CYP109A2, the remaining P450 that has not been studied in detail yet.…”

“…The substrate‐free CYP109A2 crystallized in an orthorhombic space group ( P 2 1 2 1 2 1 ), with four monomers in the asymmetric unit and a solvent content of 50%. The crystal structure was solved at 2.7 Å resolution by the molecular replacement method, using the previously determined structure of CYP109E1 (sequence similarity 45%, Protein Data Bank [PDB] entry http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5L90, ) as a search model (see Table for data collection and refinement statistics). All four CYP109A2 molecules have well‐defined electron density, except for the 15 or 16 N‐terminal residues and the C‐terminal His 6 ‐tags, for which no electron density was observed.…”

“…Most recently, our investigations led to the identification and characterization of another bacterial VD 3 ‐hydroxylase, cytochrome P450 CYP109E1 from Bacillus megaterium DSM319 . The improvement of activity and/or regio‐selectivity of the three above‐mentioned enzymes by protein engineering was supported by the knowledge of their structure–function relationships, due to determination of their crystal structures in both the open, substrate‐free and closed, substrate‐bound states .…”

Effects of Alternative Redox Partners and Oxidizing Agents on CYP154C8 Catalytic Activity and Product Distribution

“…CYPs such as CYP106A1, CYP106A2, CYP109B1, CYP109E1, CYP154C3, CYP154C5, CYP260A1, and CYP260B1, originating from bacterial sources, are known to hydroxylate steroids . CYP154C8 shows high similarity with CYP154C3 (74 %) and CYP154C5 (66 %), both of which are reported to hydroxylate steroids at C16α.…”

Characterization and structure‐guided engineering of the novel versatile terpene monooxygenase CYP109Q5 from Chondromyces apiculatus DSM436

“…The amino acid sequence identity of this novel C. apiculatus enzyme is 39% to CYP109B1, 44% to CYP109C1, 43% to CYP109C2 and 38% to CYP109D1. Studies on the steroid hydroxylating CYP109E1 from B. megaterium (36% identity to CYP109Q5) showed that this enzyme is more closely related to the steroid hydroxylase CYP106A1 in comparison with the already characterized CYP109 enzymes (Jóźwik et al ., ). Classification into a P450 family thus represents a strong evidence for a particular substrate spectrum; this is reflected by CYP109B1 and CYP109E1 that also functionalize a broad spectrum of substrates including steroids, fatty acids and terpenes (Girhard et al ., ; Putkaradze et al ., ).…”