[Adrenodoxins and their role in the cytochrome P450 systems]. / Adrenodoksiny i ikh rol' v sistemakh tsitokhroma R450.

The role of partner proteins in the formation of functional complexes in cytochrome P450 systems was investigated by means of optical biosensor technique. Kinetic constants and equilibrium dissociation constants of complexes of cytochrome CYP11A1 (P450scc) with wild-type adrenodoxin (Adx WT) and mutant forms of adrenodoxin R106D and D109R were determined using an optical biosensor. Wild-type adrenodoxin (Kd = (1.23±0.09)⋅10⁻6 M) and mutant D109R (Kd = (2.37±0.09)⋅10⁻8 M) formed complexes with cytochrome P450scc. For the R106D mutant, no complex formation was detected. To investigate the possibility of the participation of adrenodoxins and their mutant variants in the process of electron transfer as electron donors in mitochondrial cytochrome P450 systems, the electrochemical properties of these iron-sulfur proteins Adx WT and mutant forms of adrenodoxins were studied. Adx WT, mutant forms R106D and D109R have redox potentials E1/2 significantly more negative than cytochromes P450 (-579±10 mV, -590±15 mV, and -528±10 mV, respectively). These results suggest that Adx WT and mutant forms may be electron donors in the cytochrome P450 systems.

Substrate-induced modulation of protein-protein interactions within human mitochondrial cytochrome P450-dependent system.

Steroidogenesis is strictly regulated at multiple levels, as produced steroid hormones are crucial to maintain physiological functions. Cytochrome P450 enzymes are key players in adrenal steroid hormone biosynthesis and function within short redox-chains in mitochondria and endoplasmic reticulum. However, mechanisms regulating supply of reducing equivalents in the mitochondrial CYP-dependent system are not fully understood. In the present work, we aimed to estimate how the specific steroids, substrates, intermediates and products of multistep reactions modulate protein-protein interactions between adrenodoxin (Adx) and mitochondrial CYP11 s. Using the SPR technology we determined that steroid substrates affect affinity and stability of CYP11s-Adx complexes in an isoform-specific mode. In particular, cholesterol induces a 4-fold increase in the rate of CYP11A1 - Adx complex formation without significant effect on dissociation (koff decreased ∼1.5-fold), overall increasing complex affinity. At the same time steroid substrates decrease the affinity of both CYP11B1 - Adx and CYP11B2 - Adx complexes, predominantly reducing their stability (4-7 fold). This finding reveals differentiation of protein-protein interactions within the mitochondrial pool of CYPs, which have the same electron donor. The regulation of electron supply by the substrates might affect the overall steroid hormones production. Our experimental data provide further insight into protein-protein interactions within CYP-dependent redox chains involved in steroidogenesis.

Analysis of In Vivo Activity of the Bovine Cholesterol Hydroxylase/Lyase System Proteins Expressed in Escherichia coli.

The cholesterol hydroxylase/lyase (CHL) system, located in the mitochondria of the mammalian adrenal cortex cells, consists of cytochrome P450scc (CYP11A1), adrenodoxin (Adx), and adrenodoxin reductase (AdR) and performs the first stage of the steroidogenesis: AdR and Adx enable the electron transfer between NADPH and cytochrome P450scc, and P450scc catalyzes the conversion of cholesterol into pregnenolone. CHL system was reconstructed in Escherichia coli using the polycistronic plasmid pTrc99A/CHL. In E. coli cells, the recombinant proteins form the catalytically active system. CHL activity towards 22R-hydroxycholesterol was 4.0 ± 1.3 nmol pregnenolone/h per 1 mg homogenate protein. The alteration of the order of heterologous cDNAs in the expression cassette from AdR-Adx-P450scc to P450scc-Adx-AdR results in alteration of stoichiometric ratio P450scc/Adx/AdR from 1:1.45:4.2 to 1:1.67:0.98; the former ratio is more optimal for the functioning of the cytochrome P450scc. The application of modified cDNA of Adx (AdxS112W) does not increase the CHL activity; however, the introduction of the second copy of AdxS112W gene into the expression cassette increases both the expression level of АdxS112W and the CHL activity in comparison with P450scc/АdxS112W/AdR system. In vivo activity of the CHL system in bacteria is limited by the substrate uptake by bacterial cells: it varied in the range of 0.05-0.62 mg pregnenolone/l resting cell suspension per 1-day cultivation, depending on the type and concentration of permeabilizing agents in the medium. The obtained results contribute to the knowledge of CHL system functioning in living bacteria.

Novel approach to improve progesterone hydroxylation selectivity by CYP106A2 via rational design of adrenodoxin binding.

Bacterial P450s have considerable potential for biotechnological applications. The P450 CYP106A2 from Bacillus megaterium ATCC 13368 converts progesterone to several hydroxylated products that are important precursors for pharmaceutical substances. As high yields of monohydroxylated products are required for biotechnological processes, improving this conversion is of considerable interest. It has previously been shown that the binding mode of the redox partner can affect the selectivity of the progesterone hydroxylation, being more stringent in case of the Etp1 compared with Adx(4-108). Therefore, in this study we aimed to improve hydroxylation selectivity by optimizing the binding of Adx(4-108) with CYP106A2 allowing for a shorter distance between both redox centers. To change the putative binding interface of Adx(4-108) with CYP106A2, molecular docking was used to choose mutation sites for alteration. Mutants at positions Y82 and P108 of Adx were produced and investigated, and confirmed our hypothesis. Protein-protein docking, as well as conversion studies, using the mutants demonstrated that the iron-sulfur(FeS) cluster/heme distance diminished significantly, which subsequently led to an approximately 2.5-fold increase in 15ß-hydroxyprogesterone, the main product of progesterone conversion by CYP106A2.

The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species.

Mitochondrial cytochromes P450 (P450s) are responsible for important metabolic reactions, including steps involved in steroid and vitamin D metabolism. The mitochondrial P450 24A1 (CYP24A1) is responsible for deactivation of the bioactive form of vitamin D, 1,25(OH)2D3. Its function relies on formation of a P450-redox partner complex with the ferredoxin and electron donor adrenodoxin (Adx). However, very little is known about how the Adx-CYP24A1 complex forms. In this study, we report the results of solution NMR in which we monitor isotopically labeled full-length Adx as it binds CYP24A1 in complex with the P450 inhibitor clotrimazole. The NMR titration data suggested a mode for P450-Adx interactions in which formation of the complex relies on contributions from multiple recognition sites on the Adx core domain, some of which have not previously been reported. To evaluate differences among CYP24A1-Adx complexes from different mammalian species and displaying distinct regioselectivity for 1,25(OH)2D3, all bound spectra were acquired in parallel for human (carbon-23 and -24 hydroxylase), rat (carbon-24 hydroxylase), and opossum (carbon-23 hydroxylase) CYP24A1 isoforms. Binding data from a series of single and double charge-neutralizing substitutions of Adx confirmed that species-specific CYP24A1 isoforms differ in binding to Adx, providing evidence that variations in redox partner interactions correlate with P450 regioselectivity. In summary, these findings reveal that CYP24A1-Adx interactions rely on several recognition sites and that variations in CYP24A1 isoforms modulate formation of the complex, thus providing insight into the variable and complex nature of mitochondrial P450-Adx interactions.

Mitochondrial Ferredoxin Determines Vulnerability of Cells to Copper Excess.

The essential micronutrient copper is tightly regulated in organisms, as environmental exposure or homeostasis defects can cause toxicity and neurodegenerative disease. The principal target(s) of copper toxicity have not been pinpointed, but one key effect is impaired supply of iron-sulfur (FeS) clusters to the essential protein Rli1 (ABCE1). Here, to find upstream FeS biosynthesis/delivery protein(s) responsible for this, we compared copper sensitivity of yeast-overexpressing candidate targets. Overexpression of the mitochondrial ferredoxin Yah1 produced copper hyper-resistance. 55Fe turnover assays revealed that FeS integrity of Yah1 was particularly vulnerable to copper among the test proteins. Furthermore, destabilization of the FeS domain of Yah1 produced copper hypersensitivity, and YAH1 overexpression rescued Rli1 dysfunction. This copper-resistance function was conserved in the human ferredoxin, Fdx2. The data indicate that the essential mitochondrial ferredoxin is an important copper target, determining a tipping point where plentiful copper supply becomes excessive. This knowledge could help in tackling copper-related diseases.

Expression of Cholesterol Hydroxylase/Lyase System Proteins in Yeast S. cerevisiae Cells as a Self-Processing Polyprotein.

2A peptide discovered in Picornaviridae is capable of self-cleavage providing an opportunity to carry out synthesis of several proteins using one transcript. Dissociation in the 2A sequence during translation leads to the individual proteins formation. We constructed cDNA including genes of the bovine cholesterol hydroxylase/lyase (CHL) system proteins-cytochrome P450scc (CYP11A1), adrenodoxin (Adx) and adrenodoxin reductase (AdR), that are fused into a single ORF using FMDV 2A nucleotide sequences. The constructed vectors direct the expression of cDNA encoding polyprotein P450scc-2A-Adx-2A-AdR (CHL-2A) in Escherichia coli and Saccharomyces cerevisiae. The induced bacterial cells exhibit a high level of CHL-2A expression, but polyprotein is not cleaved at the FMDV sites. In yeast S. cerevisiae, the discrete proteins P450scc-2A, Adx-2A and AdR are expressed. Moreover, a significant proportion of AdR and Adx is present in a fusion Adx-2A-AdR. Thus, the first 2A linker provides an efficient cleavage of the polyprotein, while the second 2A linker demonstrates lower efficiency. Cholesterol hydroxylase/lyase activity registered in the recombinant yeast cell homogenate indicates that the catalytically active CHL system is present in these cells. Consequently, for the first time the mammalian system of cytochrome P450 has been successfully reconstructed in yeast cells through expressing the self-processing polyprotein.

Iron-sulfur cluster biogenesis and trafficking in mitochondria.

The biogenesis of iron-sulfur (Fe/S) proteins in eukaryotes is a multistage, multicompartment process that is essential for a broad range of cellular functions, including genome maintenance, protein translation, energy conversion, and the antiviral response. Genetic and cell biological studies over almost 2 decades have revealed some 30 proteins involved in the synthesis of cellular [2Fe-2S] and [4Fe-4S] clusters and their incorporation into numerous apoproteins. Mechanistic aspects of Fe/S protein biogenesis continue to be elucidated by biochemical and ultrastructural investigations. Here, we review recent developments in the pursuit of constructing a comprehensive model of Fe/S protein assembly in the mitochondrion.

Molecular Recognition in Mitochondrial Cytochromes P450 That Catalyze the Terminal Steps of Corticosteroid Biosynthesis.

The mitochondrial cytochromes P450 11B1 and P450 11B2 are responsible for the final stages of cortisol and aldosterone synthesis, respectively. Dysregulation of both enzymes has been implicated in secondary forms of hypertension. Molecular recognition of the cytochromes P450 with their corresponding redox partner is a key step in the catalytic cycle, yet the precise nature of the interaction of P450 11B1 or P450 11B2 with their proximal partner, adrenodoxin (Adx), is still unknown. Here, we obtained P450 11B1·Adx2 and P450 11B2·Adx2 complexes using the zero-length cross-linker ethyl-3-[3-(dimethylamino)propyl]carbodiimide, which formed best under low-ionic strength conditions. R-to-K mutations were introduced into the P450s at residues predicted to form salt bridges with Adx and allow cross-linking with the carbodiimide reagent. Mass spectrometric analysis of the chymotrypsin-digested ternary complexes identified seven cross-linked peptide pairs. Consistent with the electrostatic interaction of K370 in P450 11B1-WT and K366 in P450 11B2-R366K with D79 of Adx, Adx mutation L80K abolished complex formation. Using these sites of interaction as constraints, protein docking calculations based on the crystal structures of the two proteins yielded a structural model of the P450 11B1·Adx2 complex. The appositional surfaces include R/K366, K370, and K357 of P450 11B1, which interact with D79, D76, and D113 (second molecule) of Adx, respectively. Similar to P450 11B1, P450 11B2 also forms a complex with the Adx dimer via three lysine residues. We describe similarities and differences in our models of the P450 11B1·Adx2 and P450 11B2·Adx2 complexes with the structure of the P450 11A1-Adx fusion protein.

Investigating the effect of available redox protein ratios for the conversion of a steroid by a myxobacterial CYP260A1.

Since cytochromes P450 are external monooxygenases, available surrogate redox partners have been used to reconstitute the P450 activity. However, the effect of various ratios of P450s and the redox proteins have not been extensively studied so far, although different combinations of the redox partners have shown variations in substrate conversion. To address this issue, CYP260A1 was reconstituted with various ratios of adrenodoxin and adrenodoxin reductase to convert 11-deoxycorticosterone, and the products were characterized by NMR. We show the effect of the available redox protein ratios not only on the P450 catalytic activity but also on the product pattern.

A Fluorescent Readout for the Oxidation State of Electron Transporting Proteins in Cell Free Settings.

Pathways involving sequential electron transfer between multiple proteins are ubiquitous in nature. Here, we demonstrate a new class of fluorescent protein-based reporters for monitoring electron transport through such multistage cascades, specifically those involving ferredoxin-like electron transporters. We created protein fusions between mammalian Adrenodoxin (Adx) and plant Ferredoxin (Fdx) with fluorescent proteins of different colors and found that the fluorescence of such fusions is highly sensitive to the redox state of the electron transporter. The increase in fluorescence from the oxidized to the reduced state was inversely proportional to the linker length between the fusion partners. We first used our approach to quantitatively characterize electron transfer from NADPH through Adrenodoxin Reductase (AdR) to Adrenodoxin (Adx). Our data allowed us to build a detailed mathematical model of this mitochondrial electron transfer chain and validate previously proposed mechanisms. Then, we showed that an Adx-GFP fusion could serve as a sensor for the activity of bacterial Type I Cytochrome P450s (CYPs), a very large class of enzymes with important roles in biotechnology. We further showed that fluorescence of a direct fusion between CYP and GFP was sensitive to CYP activity, suggesting that our approach is applicable to an even broader class of proteins, which undergo a redox state change during their work cycle.

Association between CCDC132, FDX1 and TNFSF13 gene polymorphisms and the risk of IgA nephropathy.

AIM: Previous genome-wide association studies have identified multiple susceptibility loci for IgA nephropathy (IgAN); however, validation of these findings is still needed. METHODS: We performed a case-control study among 347 Chinese Han IgAN patients and 310 ethnicity-matched controls. Twenty-two single nucleotide polymorphisms (SNPs) were genotyped and association analysis was performed. RESULTS: We found three alleles for IgAN in patients: the allele "C" of rs2188404 in the CCDC132 gene by recessive model (odds ratio (OR), 1.65; 95% confidence interval (CI), 1.10-2.48; P = 0.014) and additive model (OR, 1.29; 95% CI, 1.03-1.61; P = 0.024) analysis, respectively, the allele "A" of rs10488764 in FDX1 gene by additive model (OR, 1.27; 95% CI, 1.00-1.61; P = 0.048) analysis, the allele "A" of rs3803800 in TNFSF13 gene by recessive model (OR, 2.05; 95% CI, 1.16-3.62; P = 0.010) and additive model (OR, 1.35; 95% CI, 1.06-1.72; P = 0.013) analysis, respectively. However, the associations between these SNPs and the risk of IgAN were not significant when adjusted for age and sex. Additionally, we found polymorphisms of rs2188404, rs10488764 and rs3803800 were correlated with urine protein (UPRO), human serum albumin (HSA), total cholesterol (TC) and Lee's pathological grades. CONCLUSION: We did not find any positive association between these SNPs and the risk of IgAN after adjustment by age and sex, but did find a significant and strong correlation with relevant clinical pathological parameters. Our study may provide a new perspective to understanding the aetiology of IgAN.

Gene expression profiling of cytochromes P450, ABC transporters and their principal transcription factors in the amygdala and prefrontal cortex of alcoholics, smokers and drug-free controls by qRT-PCR.

1. Ethanol consumption and smoking alter the expression of certain drug-metabolizing enzymes and transporters, potentially influencing the tissue-specific effects of xenobiotics. 2. Amygdala (AMG) and prefrontal cortex (PFC) are brain regions that modulate the effects of alcohol and smoking, yet little is known about the expression of cytochrome P450 enzymes (P450s) and ATP-binding cassette (ABC) transporters in these tissues. 3. Here, we describe the first study on the expression of 19 P450s, their redox partners, three ABC transporters and four related transcription factors in the AMG and PFC of smokers and alcoholics by quantitative RT-PCR. 4. CYP1A1, CYP1B1, CYP2B6, CYP2C8, CYP2C18, CYP2D6, CYP2E1, CYP2J2, CYP2S1, CYP2U1, CYP4X1, CYP46, adrenodoxin and NADPH-P450 reductase, ABCB1, ABCG2, ABCA1, and transcription factors aryl hydrocarbon receptor AhR and proliferator-activated receptor α were quantified in both areas. CYP2A6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, adrenodoxin reductase and the nuclear receptors pregnane X receptor and constitutive androstane receptor were detected but below the limit of quantification. CYP1A2 and CYP2W1 were not detected. 5. Adrenodoxin expression was elevated in all case groups over controls, and smokers showed a trend toward higher CYP1A1 and CYP1B1 expression. 6. Our study shows that most xenobiotic-metabolizing P450s and associated redox partners, transporters and transcription factors are expressed in human AMG and PFC.

Electrochemical analysis of autodisplayed adrenodoxin (Adx) on the outer membrane of E. coli.

In this work, adrenodoxin (Adx) was expressed on the outer membrane of E. coli by autodisplay and then the iron-sulfur cluster was incorporated into apo-Adx by an anaerobic reconstitution process. For the determination of the redox potentials of the iron-sulfur clusters of the autodisplayed Adx, E. coli cells with autodisplayed Adx were immobilized on a gold electrode modified with a self-assembled monolayer of mercaptoundecanoic acid (MUA). From the repeated cyclic voltammetry (CV) analysis, the E. coli (10mM HEPES buffer, pH7.0) with autodisplayed Adx showed significant changes in shape with an oxidation peak at +0.4V (vs. Ag/AgCl) and a reduction peak at -0.3V (vs. Ag/AgCl) after the reconstitution process for the incorporation of the iron-sulfur cluster. From the repeated CV analysis in the reduction and oxidation potential ranges, the iron-sulfur clusters of the autodisplayed Adx were observed to undergo reversible redox reactions via direct electron transfer to the MUA-modified gold electrode.

A recombinant CYP11B1 dependent Escherichia coli biocatalyst for selective cortisol production and optimization towards a preparative scale.

BACKGROUND: Human mitochondrial CYP11B1 catalyzes a one-step regio- and stereoselective 11ß-hydroxylation of 11-deoxycortisol yielding cortisol which constitutes not only the major human stress hormone but also represents a commercially relevant therapeutic drug due to its anti-inflammatory and immunosuppressive properties. Moreover, it is an important intermediate in the industrial production of synthetic pharmaceutical glucocorticoids. CYP11B1 thus offers a great potential for biotechnological application in large-scale synthesis of cortisol. Because of its nature as external monooxygenase, CYP11B1-dependent steroid hydroxylation requires reducing equivalents which are provided from NADPH via a redox chain, consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx). RESULTS: We established an Escherichia coli based whole-cell system for selective cortisol production from 11-deoxycortisol by recombinant co-expression of the demanded 3 proteins. For the subsequent optimization of the whole-cell activity 3 different approaches were pursued: Firstly, CYP11B1 expression was enhanced 3.3-fold to 257 nmol∗L(-1) by site-directed mutagenesis of position 23 from glycine to arginine, which was accompanied by a 2.6-fold increase in cortisol yield. Secondly, the electron transfer chain was engineered in a quantitative manner by introducing additional copies of the Adx cDNA in order to enhance Adx expression on transcriptional level. In the presence of 2 and 3 copies the initial linear conversion rate was greatly accelerated and the final product concentration was improved 1.4-fold. Thirdly, we developed a screening system for directed evolution of CYP11B1 towards higher hydroxylation activity. A culture down-scale to microtiter plates was performed and a robot-assisted, fluorescence-based conversion assay was applied for the selection of more efficient mutants from a random library. CONCLUSIONS: Under optimized conditions a maximum productivity of 0.84 g cortisol∗L(-1)∗d(-1) was achieved, which clearly shows the potential of the developed system for application in the pharmaceutical industry.

Mitochondrial targeting of cytochrome P450 (CYP) 1B1 and its role in polycyclic aromatic hydrocarbon-induced mitochondrial dysfunction.

We report that polycyclic aromatic hydrocarbon (PAH)-inducible CYP1B1 is targeted to mitochondria by sequence-specific cleavage at the N terminus by a cytosolic Ser protease (polyserase 1) to activate the cryptic internal signal. Site-directed mutagenesis, COS-7 cell transfection, and in vitro import studies in isolated mitochondria showed that a positively charged domain at residues 41-48 of human CYP1B1 is part of the mitochondrial (mt) import signal. Ala scanning mutations showed that the Ser protease cleavage site resides between residues 37 and 41 of human CYP1B1. Benzo[a]pyrene (BaP) treatment induced oxidative stress, mitochondrial respiratory defects, and mtDNA damage that was attenuated by a CYP1B1-specific inhibitor, 2,3,4,5-tetramethoxystilbene. In support, the mitochondrial CYP1B1 supported by mitochondrial ferredoxin (adrenodoxin) and ferredoxin reductase showed high aryl hydrocarbon hydroxylase activity. Administration of benzo[a]pyrene or 2,3,7,8-tetrachlorodibenzodioxin induced similar mitochondrial functional abnormalities and oxidative stress in the lungs of wild-type mice and Cyp1a1/1a2-null mice, but the effects were markedly blunted in Cyp1b1-null mice. These results confirm a role for CYP1B1 in inducing PAH-mediated mitochondrial dysfunction. The role of mitochondrial CYP1B1 was assessed using A549 lung epithelial cells stably expressing shRNA against NADPH-cytochrome P450 oxidoreductase or mitochondrial adrenodoxin. Our results not only show conservation of the endoprotease cleavage mechanism for mitochondrial import of family 1 CYPs but also reveal a direct role for mitochondrial CYP1B1 in PAH-mediated oxidative and chemical damage to mitochondria.

The novel mutation p.Trp147Arg of the steroidogenic acute regulatory protein causes classic lipoid congenital adrenal hyperplasia with adrenal insufficiency and 46,XY disorder of sex development.

BACKGROUND: The steroidogenic acute regulatory protein (StAR) is essential for steroidogenesis by mediating cholesterol transfer into mitochondria. Inactivating StAR mutations cause lipoid congenital adrenal hyperplasia. OBJECTIVE AND METHODS: To identify causative mutations in a patient presenting with adrenal failure during early infancy. The objective was to study the functional and structural consequences of the novel StAR mutation p.Trp147Arg in a Turkish patient detected in compound heterozygosity with the p.Glu169Lys mutation. RESULTS: Transient in vitro expression of the mutant proteins together with P450 side-chain cleavage enzyme, adrenodoxin, and adrenodoxin reductase yielded severely diminished cholesterol conversion of the p.Trp147Arg mutant. The previously described p.Glu169Lys mutant led to significantly lower cholesterol conversion than wild-type StAR protein. As derived from three-dimensional protein modeling, the residue W147 is stabilizing the C-terminal helix in a closed conformation hereby acting as gatekeeper of the ligand cavity of StAR. CONCLUSIONS: The novel mutation p.Trp147Arg causes primary adrenal insufficiency and complete sex reversal in the 46,XY patient. Clinical disease, in vitro studies and three-dimensional protein modeling of the mutation p.Trp147Arg underscore the relevance of this highly conserved residue for StAR protein function.

Transcriptional regulation of human ferredoxin 1 in ovarian granulosa cells.

Ferredoxin 1 (FDX1; adrenodoxin) is an iron-sulfur protein that is involved in various metabolic processes, including steroid hormone synthesis in mammalian tissues. We investigated the transcriptional regulation of FDX1 in ovarian granulosa cells. Previously, we reported that the NR5A family, including steroidogenic factor-1 (SF-1) and liver receptor homolog-1 could induce differentiation of human mesenchymal stem cells (hMSCs) into steroidogenic cells. A ChIP assay showed that SF-1 could bind to the FDX1 promoter in differentiated hMSCs. Luciferase reporter assays showed that transcription of FDX1 was synergistically activated by the NR5A family and 8Br-cAMP treatment through two SF-1 binding sites and a CRE-like sequence in a human ovarian granulosa cell line, KGN. Knockdown of FDX1 attenuated progesterone production in KGN cells. These results indicate transcription of FDX1 is regulated by the NR5A family and cAMP signaling, and participates in steroid hormone production in ovarian granulosa cells.

Application of a new versatile electron transfer system for cytochrome P450-based Escherichia coli whole-cell bioconversions.

Cytochromes P450 monooxygenases are highly interesting biocatalysts for biotechnological applications, since they perform a diversity of reactions on a broad range of organic molecules. Nevertheless, the application of cytochromes P450 is limited compared to other enzymes mainly because of the necessity of a functional redox chain to transfer electrons from NAD(P)H to the monooxygenase. In this study, we established a novel robust redox chain based on adrenodoxin, which can deliver electrons to mitochondrial, bacterial and microsomal P450s. The natural membrane-associated reductase of adrenodoxin was replaced by the soluble Escherichia coli reductase. We could demonstrate for the first time that this reductase can transfer electrons to adrenodoxin. In the first step, the electron transfer properties and the potential of this new system were investigated in vitro, and in the second step, an efficient E. coli whole-cell system using CYP264A1 from Sorangium cellulosum So ce56 was developed. It could be demonstrated that this novel redox chain leads to an initial conversion rate of 55 µM/h, which was 52 % higher compared to the 36 µM/h of the redox chain containing adrenodoxin reductase. Moreover, we optimized the whole-cell biotransformation system by a detailed investigation of the effects of different media. Finally, we are able to demonstrate that the new system is generally applicable to other cytochromes P450 by combining it with the biotechnologically important steroid hydroxylase CYP106A2 from Bacillus megaterium.

A new Bacillus megaterium whole-cell catalyst for the hydroxylation of the pentacyclic triterpene 11-keto-ß-boswellic acid (KBA) based on a recombinant cytochrome P450 system.

The use of cytochromes P450 for the regio- and stereoselective hydroxylation of non-activated carbon atoms in biotechnological applications reflects an efficient and cost-effective alternative in comparison to classical organic chemistry. The prokaryotic cytochrome P450 CYP106A2 from Bacillus megaterium ATCC 13368 hydroxylates a variety of 3-oxo-Δ4 steroids and recently it was identified to carry out a one-step regioselective allylic hydroxylation of the diterpene abietic acid. The anti-inflammatory pentacyclic triterpene 11-keto-ß-boswellic acid (KBA) was found to be a further substrate of CYP106A2, being the first report of a pentacyclic triterpene conversion by a prokaryotic P450. The reaction products were analyzed by HPLC and the corresponding kinetic parameters were investigated. Structure determination of the main product by NMR revealed a 15α-hydroxylation of this substrate. In order to overcome the inability of a recombinant P450 whole-cell system in E. coli for the uptake of acids with terpene structure, we developed for the first time an expression system for cytochromes P450 in B. megaterium (strains MS941 and ATCC 13368). Interestingly, CYP106A2 was only successfully expressed in the plasmid-less B. megaterium strain MS941 but not in ATCC13368. This recombinant system, with the co-expressed heterologous redox chain of the P450, bovine adrenodoxin reductase (AdR), and bovine adrenodoxin (Adx), was applied for the whole-cell conversion of KBA. The formation of 15α-hydroxy-KBA was increased 15-fold in comparison with the naturally CYP106A2-expressing B. megaterium strain ATCC 13368.