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

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 16ß-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.

Resurrection and characterization of ancestral CYP11A1 enzymes.

Mitochondrial cytochromes P450 presumably originated from a common microsomal P450 ancestor. However, it is still unknown how ancient mitochondrial P450s were able to retain their oxygenase function following relocation to the mitochondrial matrix and later emerged as enzymes specialized for steroid hormone biosynthesis in vertebrates. Here, we used the approach of ancestral sequence reconstruction (ASR) to resurrect ancient CYP11A1 enzymes and characterize their unique biochemical properties. Two ancestral CYP11A1 variants, CYP11A_Mammal_N101 and CYP11A_N1, as well as an extant bovine form were recombinantly expressed and purified to homogeneity. All enzymes showed characteristic P450 spectral properties and were able to convert cholesterol as well as other sterol substrates to pregnenolone, yet with different specificities. The vertebrate CYP11A_N1 ancestor preferred the cholesterol precursor, desmosterol, as substrate suggesting a convergent evolution of early cholesterol metabolism and CYP11A1 enzymes. Both ancestors were able to withstand increased levels of hydrogen peroxide but only the ancestor CYP11A_N1 showed increased thermostability (˜ 25 °C increase in T50 ) compared with the extant CYP11A1. The extraordinary robustness of ancient mitochondrial P450s, as demonstrated for CYP11A_N1, may have allowed them to stay active when presented with poorly compatible electron transfer proteins and resulting harmful ROS in the new environment of the mitochondrial matrix. To the best of our knowledge, this work represents the first study that describes the resurrection of ancient mitochondrial P450 enzymes. The results will help to understand and gain fundamental functional insights into the evolutionary origins of steroid hormone biosynthesis in animals.

Improvement of the 25-hydroxyvitamin D3 production in a CYP109A2-expressing Bacillus megaterium system.

Calcifediol (25(OH)VD3) is a physiologically very important vitamin D3 metabolite and of high pharmaceutical importance, due to its potential for treating not only vitamin D3 deficiencies but also coronary diseases and cancer. Previously, we established a whole-cell Bacillus megaterium-based system using the cytochrome P450 CYP109A2 for the biotransformation of vitamin D3 into its metabolite 25-hydroxyvitamin D3. In this study, we demonstrate the importance of the region between amino acids T103 and A106 for the catalytic activity of CYP109A2 towards vitamin D3 as a substrate. In order to increase the productivity of the system, reaction conditions (xylose, vitamin D3, saponin, 2-hydroxypropyl-ß-cyclodextrin) were optimized for the in vivo production of 25-hydroxyvitamin D3. With cells producing the T103A mutant, a productivity of 282.7 mg/L/48 h was achieved under the optimized conditions. This value is two times higher than that obtained in the control reaction with the wild-type enzyme in this study and five times higher than that obtained in a previous study.

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.

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).

Identification of a new plasmid-encoded cytochrome P450 CYP107DY1 from Bacillus megaterium with a catalytic activity towards mevastatin.

In the current work, we describe the identification and characterization of the first plasmid-encoded P450 (CYP107DY1) from a Bacillus species. The recombinant CYP107DY1 exhibits characteristic P450 absolute and reduced CO-bound difference spectra. Reconstitution with different redox systems revealed the autologous one, consisting of BmCPR and Fdx2, as the most effective one. Screening of a library of 18 pharmaceutically relevant compounds displayed activity towards mevastatin to produce pravastatin. Pravastatin is an important therapeutic drug to treat hypercholesterolemia, which was described to be produced by oxyfunctionlization of mevastatin (compactin) by members of CYP105 family. The hydroxylation at C6 of mevastatin was also suggested by docking this compound into a computer model created for CYP107DY1. Moreover, in view of the biotechnological application, CYP107DY1 as well as its redox partners (BmCPR and Fdx2) were successfully utilized to establish an E. coli based whole-cell system for an efficient biotransformation of mevastatin. The in vitro and in vivo application of the CYP07DY1 also offers the possibility for the screening of more substrates, which could open up further biotechnological usage of this enzyme.

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.

Conserved gating elements in TRPC4 and TRPC5 channels.

TRPC4 and TRPC5 proteins share 65% amino acid sequence identity and form Ca(2+)-permeable nonselective cation channels. They are activated by stimulation of receptors coupled to the phosphoinositide signaling cascade. Replacing a conserved glycine residue within the cytosolic S4-S5 linker of both proteins by a serine residue forces the channels into an open conformation. Expression of the TRPC4G503S and TRPC5G504S mutants causes cell death, which could be prevented by buffering the Ca(2+) of the culture medium. Current-voltage relationships of the TRPC4G503S and TRPC5G504S mutant ion channels resemble that of fully activated TRPC4 and TRPC5 wild-type channels, respectively. Modeling the structure of the transmembrane domains and the pore region (S4-S6) of TRPC4 predicts a conserved serine residue within the C-terminal sequence of the predicted S6 helix as a potential interaction site. Introduction of a second mutation (S623A) into TRPC4G503S suppressed the constitutive activation and partially rescued its function. These results indicate that the S4-S5 linker is a critical constituent of TRPC4/C5 channel gating and that disturbance of its sequence allows channel opening independent of any sensor domain.