Investigation of cytochromes P450 in myxobacteria: Excavation of cytochromes P450 from the genome of Sorangium cellulosum So ce56

AbstractThe exploitation of cytochromes P450 for novel biotechnological application and for the investigation of their physiological function is of great scientific interest in this post genomic era, where an extraordinary biodiversity of P450 genes has been derived from all forms of life. The study of P450s in the myxobacterium Sorangium cellulosum strain So ce56, the producer of novel secondary metabolites of pharmaceutical interest is the research topic, in which we were engaged since the beginning of its genome sequencing project. We herein disclosed the cytochrome P450 complements (CYPomes) of spore-forming myxobacterial species, Stigmatella aurantiaca DW4/3-1, Haliangium ochraceum DSM 14365 and Myxococcus xanthus DK1622, and their potential pharmaceutical significance has been discussed

Hemin rescues adrenodoxin, heme a and cytochrome oxidase activity in frataxin-deficient oligodendroglioma cells

AbstractMutations in the frataxin gene cause neurodegeneration and demyelination in Friedreich's ataxia. We showed earlier that frataxin deficiency causes primary iron–sulfur cluster defects, and later causes defects in heme and cytochrome c hemoprotein levels. Iron–sulfur (Fe/S) clusters are required in two enzymes of heme biosynthesis in humans i.e. in ferrochelatase and adrenodoxin. However, decreases in ferrochelatase activity have not been observed in frataxin-deficient HeLa cells or patient lymphoblasts. We knocked down frataxin in oligodendroglioma cells using siRNA, which produced significant defects in the activity of the Fe/S cluster enzymes adrenodoxin and aconitase, the adrenodoxin product heme a, and cytochrome oxidase, for which heme a serves as a prosthetic group. Exogenous hemin produced a significant rescue of adrenodoxin, aconitase, heme a levels and cytochrome oxidase activity. Thus hemin rescues iron–sulfur cluster defects that are the result of frataxin-deficiency, perhaps as a consequence of increasing the pool of bioavailable iron, and thus should be more fully tested for beneficial effects in Friedreich's ataxia models

A CYP21A2 based whole-cell system in Escherichia coli for the biotechnological production of premedrol

Additional file 4: Fig. S4. In vitro conversion of medrane with the redox systems AdR/Adx/CYP21A2 or arh1/Adx/CYP21A2 with either NADH or NADPH. 400 ÎźM Medrane was converted in a reconstituted in vitro assay with Adx based redox systems containing AdR or arh1 as reductase. To each system either NADH or NADPH was added to verify the ability of arh1 to receive electrons from NADH. AdR served as a negative control. All values represent the mean of triplicates with the respective standard deviation

A CYPome-wide study reveals new potential players in the pathogenesis of Parkinson's disease

Genetic and environmental factors lead to the manifestation of Parkinson's disease (PD) but related mechanisms are only rudimentarily understood. Cytochromes P450 (P450s) are involved in the biotransformation of toxic compounds and in many physiological processes and thus predestinated to be involved in PD. However, so far only SNPs (single nucleotide polymorphisms) in CYP2D6 and CYP2E1 have been associated with the susceptibility of PD. Our aim was to evaluate the role of all 57 human P450s and their redox partners for the etiology and pathophysiology of PD and to identify novel potential players which may lead to the identification of new biomarkers and to a causative treatment of PD. The PPMI (Parkinson's Progression Markers Initiative) database was used to extract the gene sequences of all 57 P450s and their three redox partners to analyze the association of SNPs with the occurrence of PD. Applying statistical analyses of the data, corresponding odds ratios (OR) and confidence intervals (CI) were calculated. We identified SNPs significantly over-represented in patients with a genetic predisposition for PD (GPD patients) or in idiopathic PD (IPD patients) compared to HC (healthy controls). Xenobiotic-metabolizing P450s show a significant accumulation of SNPs in PD patients compared with HC supporting the role of toxic compounds in the pathogenesis of PD. Moreover, SNPs with high OR values (>5) in P450s catalyzing the degradation of cholesterol (CYP46A1, CY7B1, CYP39A1) indicate a prominent role of cholesterol metabolism in the brain for PD risk. Finally, P450s participating in the metabolism of eicosanoids show a strong over-representation of SNPs in PD patients underlining the effect of inflammation on the pathogenesis of PD. Also, the redox partners of P450 show SNPs with OR > 5 in PD patients. Taken together, we demonstrate that SNPs in 26 out of 57 P450s are at least 5-fold over-represented in PD patients suggesting these P450s as new potential players in the pathogenesis of PD. For the first time exceptionally high OR values (up to 12.9) were found. This will lead to deeper insight into the origin and development of PD and may be applied to develop novel strategies for a causative treatment of this disease.The work was supported by a research grant from the “Dr. Rolf M. Schwiete Stiftung” Mannheim/Germany

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 11β-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

Structural characterization of CYP260A1 from Sorangium cellulosum to investigate the 1α‐hydroxylation of a mineralocorticoid

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135350/1/feb212479.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135350/2/feb212479_am.pd

Structural comparison of the cytochrome P450 enzymes CYP106A1 and CYP106A2 provides insight into their differences in steroid conversion

Understanding the structural basis of the selectivity of steroid hydroxylation requires detailed structural and functional investigations on various steroid hydroxylases with different selectivities, such as the bacterial cytochrome P450 enzymes. Here, the crystal structure of the cytochrome P450 CYP106A1 from Priestia megaterium was solved. CYP106A1 exhibits a rare additional structural motif of a cytochrome P450, a sixth β-sheet. The protein was found in different unusual conformations corresponding to both open and closed forms even when crystallized without any known substrate. The structural comparison of CYP106A1 with the previously investigated CYP106A2, including docking studies for both isoforms with the substrate cortisol, reveals a completely different orientation of the steroid molecule in the active sites. This distinction convincingly explains the experimentally observed differences in substrate conversion and product formation by the two enzymes

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 11β-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

New aspects of electron transfer revealed by the crystal structure of a truncated bovine adrenodoxin, Adx(4–108)

AbstractBackground: Adrenodoxin (Adx) is a [2Fe–2S] ferredoxin involved in steroid hormone biosynthesis in the adrenal gland mitochondrial matrix of mammals. Adx is a small soluble protein that transfers electrons from adrenodoxin reductase (AR) to different cytochrome P450 isoforms where they are consumed in hydroxylation reactions. A crystallographic study of Adx is expected to reveal the structural basis for an important electron transfer reaction mediated by a vertebrate [2Fe–2S] ferredoxin.Results: The crystal structure of a truncated bovine adrenodoxin, Adx(4–108), was determined at 1.85 å resolution and refined to a crystallographic R value of 0.195. The structure was determined using multiple wavelength anomalous dispersion phasing techniques, making use of the iron atoms in the [2Fe–2S] cluster of the protein. The protein displays the compact (α+β) fold typical for [2Fe–2S] ferredoxins. The polypeptide chain is organized into a large core domain and a smaller interaction domain which comprises 35 residues, including all those previously determined to be involved in binding to AR and cytochrome P450. A small interdomain motion is observed as a structural difference between the two independent molecules in the asymmetric unit of the crystal. Charged residues of Adx(4–108) are clustered to yield a strikingly asymmetric electric potential of the protein molecule.Conclusions: The crystal structure of Adx(4–108) provides the first detailed description of a vertebrate [2Fe–2S] ferredoxin and serves to explain a large body of biochemical studies in terms of a three-dimensional structure. The structure suggests how a change in the redox state of the [2Fe–2S] cluster may be coupled to a domain motion of the protein. It seems likely that the clearly asymmetric charge distribution on the surface of Adx(4–108) and the resulting strong molecular dipole are involved in electrostatic steering of the interactions with AR and cytochrome P450