CYP109E1 is a novel versatile statin and terpene oxidase from Bacillus megaterium.

CYP109E1 is a cytochrome P450 monooxygenase from Bacillus megaterium with a hydroxylation activity for testosterone and vitamin D3. This study reports the screening of a focused library of statins, terpene-derived and steroidal compounds to explore the substrate spectrum of this enzyme. Catalytic activity of CYP109E1 towards the statin drug-precursor compactin and the prodrugs lovastatin and simvastatin as well as biotechnologically relevant terpene compounds including ionones, nootkatone, isolongifolen-9-one, damascones, and ß-damascenone was found in vitro. The novel substrates induced a type I spin-shift upon binding to P450 and thus permitted to determine dissociation constants. For the identification of conversion products by NMR spectroscopy, a B. megaterium whole-cell system was applied. NMR analysis revealed for the first time the ability of CYP109E1 to catalyze an industrially highly important reaction, the production of pravastatin from compactin, as well as regioselective oxidations generating drug metabolites (6'ß-hydroxy-lovastatin, 3'α-hydroxy-simvastatin, and 4″-hydroxy-simvastatin) and valuable terpene derivatives (3-hydroxy-α-ionone, 4-hydroxy-ß-ionone, 11,12-epoxy-nootkatone, 4(R)-hydroxy-isolongifolen-9-one, 3-hydroxy-α-damascone, 4-hydroxy-ß-damascone, and 3,4-epoxy-ß-damascone). Besides that, a novel compound, 2-hydroxy-ß-damascenone, produced by CYP109E1 was identified. Docking calculations using the crystal structure of CYP109E1 rationalized the experimentally observed regioselective hydroxylation and identified important amino acid residues for statin and terpene binding.

A new cytochrome P450 system from Bacillus megaterium DSM319 for the hydroxylation of 11-keto-ß-boswellic acid (KBA).

In the genome of Bacillus megaterium DSM319, a strain who has recently been sequenced to fully exploit its potential for biotechnological purposes, we identified a gene encoding the cytochrome P450 CYP106A1 as well as genes encoding potential redox partners of CYP106A1. We cloned, expressed, and purified CYP106A1 and five potential autologous redox partners, one flavodoxin and four ferredoxins. The flavodoxin and three ferredoxins were able to support the activity of CYP106A1 displaying the first cloned natural redox partners of a cytochrome P450 from B. megaterium. The CYP106A1 system was able to convert the pentacyclic triterpene 11-keto-ß-boswellic acid (KBA) belonging to the main bioactive constituents of Boswellia serrata gum resin extracts, which are used to treat inflammatory disorders and arthritic diseases. In order to provide sufficient amounts of the KBA products to characterize them structurally by NMR spectroscopy, recombinant whole-cell biocatalysts were constructed based on B. megaterium MS941. The main product has been identified as 7ß-hydroxy-KBA, while the side product (∼20%) was shown to be a mixture of 7ß,15α-dihydroxy-KBA and 15α-hydroxy-KBA. Without further optimization 560.7 mg l⁻¹ day⁻¹ of the main product, 7ß-hydroxy-KBA, could be obtained thus providing a suitable starting point for the efficient production of modified KBA by chemical tailoring to produce novel KBA derivatives with increased bioavailability and this way more efficient drugs.

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

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 D3 (VD3 ) metabolites are of pharmaceutical importance and are applied for the treatment of VD3 deficiency and other disorders. However, the chemical synthesis of VD3 derivatives shows low specificity and low yields. In this study, cytochrome P450 CYP109A2 from Bacillus megaterium DSM319 was expressed, purified, and shown to oxidize VD3 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 VD3 . 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 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 VD3 binding as the related VD3 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 VD3 hydroxylation. DATABASE: The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession code 5OFQ (substrate-free CYP109A2). 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.

Characterization of cytochrome P450 CYP109E1 from Bacillus megaterium as a novel vitamin D3 hydroxylase.

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), VD3 was 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 VD3 at 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)2VD3 by CYP109E1 is derived from VD3 via 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 VD3 compared with the wild type activity, resulting in an approximately 2-fold increase of 25(OH)VD3 production (45mgl-1day-1) compared to wild type (24.5mgl-1day-1).

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

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 16ß. 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. DATABASE: The 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). ENZYMES: Cytochrome P450 monooxygenase CYP109E1, EC 1.14.14.1, UniProt ID: D5DKI8, Adrenodoxin reductase EC 1.18.1.6.