Roles of Ferric Peroxide Anion Intermediates (Fe3+O2 -, Compound 0) in Cytochrome P450 19A1 Steroid Aromatization and a Cytochrome P450 2B4 Secosteroid Oxidation Model.

Cytochrome P450 (P450, CYP) 19A1 is the steroid aromatase, the enzyme responsible for the 3-step conversion of androgens (androstenedione or testosterone) to estrogens. The final step is C-C bond scission (removing the 19-oxo group as formic acid) that proceeds via a historically controversial reaction mechanism. The two competing mechanistic possibilities involve a ferric peroxide anion (Fe3+O2 -, Compound 0) and a perferryl oxy species (FeO3+, Compound I). One approach to discern the role of each species in the reaction is with the use of oxygen-18 labeling, i.e., from 18O2 and H2 18O of the reaction product formic acid. We applied this approach, using several technical improvements, to study the deformylation of 19-oxo-androstenedione by human P450 19A1 and of a model secosteroid, 3-oxodecaline-4-ene-10-carboxaldehyde (ODEC), by rabbit P450 2B4. Both aldehyde substrates were sensitive to non-enzymatic acid-catalyzed deformylation, yielding 19-norsteroids, and conditions were established to avoid issues with artifactual generation of formic acid. The Compound 0 reaction pathway predominated (i.e., Fe3+O2 -) in both P450 19A1 oxidation of 19-oxo-androstenedione and P450 2B4 oxidation of ODEC. The P450 19A1 results contrast with our prior conclusions (J. Am. Chem. Soc. 2014, 136, 15016-16025), attributed to several technical modifications.

In-depth gas chromatography/tandem mass spectrometry fragmentation analysis of formestane and evaluation of mass spectral discrimination of isomeric 3-keto-4-ene hydroxy steroids.

RATIONALE: The aromatase inhibitor formestane (4-hydroxyandrost-4-ene-3,17-dione) is included in the World Anti-Doping Agency's List of Prohibited Substances in Sport. However, it also occurs endogenously as do its 2-, 6- and 11-hydroxy isomers. The aim of this study is to distinguish the different isomers using gas chromatography/electron ionization mass spectrometry (GC/EI-MS) for enhanced confidence in detection and selectivity for determination. METHODS: Established derivatization protocols to introduce [2 H9 ]TMS were followed to generate perdeuterotrimethylsilylated and mixed deuterated derivatives for nine different hydroxy steroids, all with 3-keto-4-ene structure. Formestane was additionally labelled with H2 18 O to obtain derivatives doubly labelled with [2 H9 ]TMS and 18 O. GC/EI-MS spectra of labelled and unlabelled TMS derivatives were compared. Proposals for the generation of fragment ions were substantiated by high-resolution MS (GC/QTOFMS) and tandem mass spectrometry (MS/MS) experiments. RESULTS: Subclass-specific fragment ions include m/z 319 for the 6-hydroxy and m/z 219 for the 11-hydroxy compounds. Ions at m/z 415, 356, 341, 313, 269 and 267 were indicative for the 2- and 4-hydroxy compounds. For their discrimination the transition m/z 503 → 269 was selective for formestane. In 2-, 4- and 6-hydroxy steroids loss of a TMSO radical takes place as cleavage of a TMS-derived methyl radical and a neutral loss of (CH3 )2 SiO. Further common fragments were also elucidated. CONCLUSIONS: With the help of stable isotope labelling, the structures of postulated diagnostic fragment ions for the different steroidal subclasses were elucidated. 18 O-labelling of the other compounds will be addressed in future studies to substantiate the obtained findings. To increase method sensitivity MS3 may be suitable in future bioanalytical applications requiring discrimination of the 2- and 4-hydroxy compounds.

Biotransformation of androst-4-ene-3,17-dione and nandrolone decanoate by genera of Aspergillus and Fusarium.

The ability of five fungal species belonging to two genera of Aspergillus and Fusarium has been examined in the microbial transformation of androst-4-ene-3, 17-dione (AD). Furthermore, the biotransformation of nandrolone decanoate (2) by F. fujikuroi has been studied. AD (1) was converted by cultures of Aspergillus sp. PTCC 5266 to form 11α-hydroxy-AD (3) as the only product, with a yield of 86% in 3 days. Moreover, two hydroxylated metabolites 11α-hydroxy-AD (3, 65%) and 7ß-hydroxy-AD (4; 18%) were isolated in biotransformation of AD by A. nidulans. On the other hand, it was metabolized by F. oxysporum to produce 14α-hydroxy-AD (5; 38%) and testosterone (6; 12%). Microbial transformation of AD by F. solani led to the production of 11α-hydroxy-AD (3; 54%) and testosterone (6; 14%). AD was reduced at the 17-position by F. fujikuroi to produce testosterone in the yield of 42%. Finally, nandrolone decanoate was transformed by F. fujikuroi via hydrolysis and oxidation at the 17-position to produce two metabolites namely 17ß-hydroxyestr-4-en-3-one (7, 25.4%) and estr-4-en-3,17-dione (8, 33%), respectively. The all metabolites were purified and subsequently identified based on their spectra data analysis and comparing them to the literature data.

Microbial Modifications of Androstane and Androstene Steroids by Penicillium vinaceum.

The biotransformation of steroid compounds is a promising, environmentally friendly route to new pharmaceuticals and hormones. One of the reaction types common in the metabolic fate of steroids is Baeyer-Villiger oxidation, which in the case of cyclic ketones, such as steroids, leads to lactones. Fungal enzymes catalyzing this reaction, Baeyer-Villiger monooxygenases (BVMOs), have been shown to possess broad substrate scope, selectivity, and catalytic performance competitive to chemical oxidation, being far more environmentally green. This study covers the biotransformation of a series of androstane steroids (epiandrosterone and androsterone) and androstene steroids (progesterone, pregnenolone, dehydroepiandrosterone, androstenedione, 19-OH-androstenedione, testosterone, and 19-nortestosterone) by the cultures of filamentous fungus Penicillium vinaceum AM110. The transformation was monitored by GC and the resulting products were identified on the basis of chromatographic and spectral data. The investigated fungus carries out effective Baeyer-Villiger oxidation of the substrates. Interestingly, introduction of the 19-OH group into androstenedione skeleton has significant inhibitory effect on the BVMO activity, as the 10-day transformation leaves half of the 19-OH-androstenedione unreacted. The metabolic fate of epiandrosterone and androsterone, the only 5α-saturated substrates among the investigated compounds, is more complicated. The transformation of these two substrates combined with time course monitoring revealed that each substrate is converted into three products, corresponding to oxidation at C-3 and C-17, with different time profiles and yields.

Genome-wide response on phytosterol in 9-hydroxyandrostenedione-producing strain of Mycobacterium sp. VKM Ac-1817D.

BACKGROUND: Aerobic side chain degradation of phytosterols by actinobacteria is the basis for the industrial production of androstane steroids which are the starting materials for the synthesis of steroid hormones. A native strain of Mycobacterium sp. VKM Ac-1817D effectively produces 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) from phytosterol, but also is capable of slow steroid core degradation. However, the set of the genes with products that are involved in phytosterol oxidation, their organisation and regulation remain poorly understood. RESULTS: High-throughput sequencing of the global transcriptomes of the Mycobacterium sp. VKM Ac-1817D cultures grown with or without phytosterol was carried out. In the presence of phytosterol, the expression of 260 genes including those related to steroid catabolism pathways significantly increased. Two of the five genes encoding the oxygenase unit of 3-ketosteroid-9α-hydroxylase (kshA) were highly up-regulated in response to phytosterol (55- and 25-fold, respectively) as well as one of the two genes encoding its reductase subunit (kshB) (40-fold). Only one of the five putative genes encoding 3-ketosteroid-∆1-dehydrogenase (KstD_1) was up-regulated in the presence of phytosterol (61-fold), but several substitutions in the conservative positions of its product were revealed. Among the genes over-expressed in the presence of phytosterol, several dozen genes did not possess binding sites for the known regulatory factors of steroid catabolism. In the promoter regions of these genes, a regularly occurring palindromic motif was revealed. The orthologue of TetR-family transcription regulator gene Rv0767c of M. tuberculosis was identified in Mycobacterium sp. VKM Ac-1817D as G155_05115. CONCLUSIONS: High expression levels of the genes related to the sterol side chain degradation and steroid 9α-hydroxylation in combination with possible defects in KstD_1 may contribute to effective 9α-hydroxyandrost-4-ene-3,17-dione accumulation from phytosterol provided by this biotechnologically relevant strain. The TetR-family transcription regulator gene G155_05115 presumably associated with the regulation of steroid catabolism. The results are of significance for the improvement of biocatalytic features of the microbial strains for the steroid industry.

Kinetic processivity of the two-step oxidations of progesterone and pregnenolone to androgens by human cytochrome P450 17A1.

Cytochrome P450 (P450, CYP) 17A1 plays a critical role in steroid metabolism, catalyzing both the 17α-hydroxylation of pregnenolone and progesterone and the subsequent 17α,20-lyase reactions to form dehydroepiandrosterone (DHEA) and androstenedione (Andro), respectively, critical for generating glucocorticoids and androgens. Human P450 17A1 reaction rates examined are enhanced by the accessory protein cytochrome b5 (b5), but the exact role of b5 in P450 17A1-catalyzed reactions is unclear as are several details of these reactions. Here, we examined in detail the processivity of the 17α-hydroxylation and lyase steps. b5 did not enhance reaction rates by decreasing the koff rates of any of the steroids. Steroid binding to P450 17A1 was more complex than a simple two-state system. Pre-steady-state experiments indicated lag phases for Andro production from progesterone and for DHEA from pregnenolone, indicating a distributive character of the enzyme. However, we observed processivity in pregnenolone/DHEA pulse-chase experiments. (S)-Orteronel was three times more inhibitory toward the conversion of 17α-hydroxypregnenolone to DHEA than toward the 17α-hydroxylation of pregnenolone. IC50 values for (S)-orteronel were identical for blocking DHEA formation from pregnenolone and for 17α-hydroxylation, suggestive of processivity. Global kinetic modeling helped assign sets of rate constants for individual or groups of reactions, indicating that human P450 17A1 is an inherently distributive enzyme but that some processivity is present, i.e. some of the 17α-OH pregnenolone formed from pregnenolone did not dissociate from P450 17A1 before conversion to DHEA. Our results also suggest multiple conformations of P450 17A1, as previously proposed on the basis of NMR spectroscopy and X-ray crystallography.

A Dehydrogenase Dual Hydrogen Abstraction Mechanism Promotes Estrogen Biosynthesis: Can We Expand the Functional Annotation of the Aromatase Enzyme?

Cytochrome P450 (CYP450) enzymes are involved in the metabolism of exogenous compounds and in the synthesis of signaling molecules. Among the latter, human aromatase (HA) promotes estrogen biosynthesis, which is a key pharmacological target against breast cancers. After decades of debate, interest in gaining a comprehensive picture of HA catalysis has been renewed by the recent discovery that compound I (Cpd I) is the reactive species of the peculiar aromatization step. Herein, for the first time, a complete atomic-level picture of all controversial steps of estrogen biosynthesis is presented. By performing cumulative quantum-classical molecular dynamics and metadynamics simulations of about 180 ps, it is revealed that the most likely enzymatic path relies on three factors: 1) androstenedione enolization and compound 0 (Cpd 0) formation through a proton network mediated by Asp309; 2) subsequent formation of Cpd I, upon rearrangement of the Asp309 side chain and the establishment of a proton network involving Asp309 and Thr310; and 3) after two hydroxylation reactions, 19,19-gem-diol is converted into estrone by Cpd I, through an uncommon dehydrogenase-like dual hydrogen abstraction mechanism. As a result, HA performs estrogen biosynthesis by merging hydroxylase with dehydrogenase activity, which suggests that the need to perform complex chemical transformations led nature to engineer HA, and possibly other steroidogenic CYP450s, by expanding its range of functions to achieve an optimal catalytic efficiency.

Engineering phytosterol transport system in Mycobacterium sp. strain MS136 enhances production of 9α-hydroxy-4-androstene-3,17-dione.

OBJECTIVES: To enhance the yield of 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) from phytosterols, a phytosterol transport system was constructed in Mycobacterium sp. strain MS136. RESULTS: 9-OHAD can be produced via the controlled degradation of phytosterols by mycobacteria. This involves an active transport process that requires trans-membrane proteins and ATP. A phytosterol transport system from Mycobacterium tuberculosis H37Rv was constructed in Mycobacterium sp. strain MS136 by co-expression of an energy-related gene, mceG, and two integrated membrane protein genes, yrbE4A and yrbE4B. The resultant of the Mycobacterium sp. strain MS136-GAB gave 5.7 g 9-OHAD l-1, which was a 20% increase over 4.7 g l-1 by the wild-type strain. The yield of 9-OHAD was increased to 6.0 g l-1 by optimization of fermentation conditions, when 13 g phytosterols l-1 were fermented for 84 h in 30 ml biotransformation medium in shake flasks. CONCLUSIONS: Phytosterol transport system plays an active role in the uptake and transport of sterols, cloning of the system improved the mass transfer of phytosterols and increased the production of 9-OHAD.

Overexpression of cytochrome p450 125 in Mycobacterium: a rational strategy in the promotion of phytosterol biotransformation.

Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are generally produced by the biotransformation of phytosterols in Mycobacterium. The AD (D) production increases when the strain has high NAD+/NADH ratio. To enhance the AD (D) production in Mycobacterium neoaurum TCCC 11978 (MNR M3), a rational strategy was developed through overexpression of a gene involved in the phytosterol degradation pathway; NAD+ was generated as well. Proteomic analysis of MNR cultured with and without phytosterols showed that the steroid C27-monooxygenase (Cyp125-3), which performs sequential oxidations of the sterol side chain at the C27 position and has the oxidative cofactor of NAD+ generated, played an important role in the phytosterol biotransformation process of MNR M3. To improve the productivity of AD (D), the cyp125-3 gene was overexpressed in MNR M3. The specific activity of Cyp125-3 in the recombinant strain MNR M3C3 was improved by 22% than that in MNR M3. The NAD+/NADH ratio in MNR M3C3 was 131% higher than that in the parent strain. During phytosterol biotransformation, the conversion of sterols increased from 84 to 96%, and the yield of AD (D) by MNR M3C3 was increased by approximately 18% for 96 h fermentation. This rational strain modification strategy may also be applied to develop strains with important application values for efficient production of cofactor-dependent metabolites.

Δ4-3-ketosteroids as a new class of substrates for the cytosolic sulfotransferases.

Cytosolic sulfotransferase (SULT)-mediated sulfation is generally known to involve the transfer of a sulfonate group from the active sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), to a hydroxyl group or an amino group of a substrate compound. We report here that human SULT2A1, in addition to being able to sulfate dehydroepiandrosterone (DHEA) and other hydroxysteroids, could also catalyze the sulfation of Δ4-3-ketosteroids, which carry no hydroxyl groups in their chemical structure. Among a panel of Δ4-3-ketosteroids tested as substrates, 4-androstene-3,17-dione and progesterone were found to be sulfated by SULT2A1. Mass spectrometry analysis and structural modeling supported a reaction mechanism which involves the isomerization of Δ4-3-ketosteroids from the keto form to an enol form, prior to being subjected to sulfation. Results derived from this study suggested a potential role of SULT2A1 as a Δ4-3-ketosteroid sulfotransferase in steroid metabolism.

Exploring new chemical functionalities to improve aromatase inhibition of steroids.

In this work, new potent steroidal aromatase inhibitors both in microsomes and in breast cancer cells have been found. The synthesis of the 3,4-(ethylenedioxy)androsta-3,5-dien-17-one (12), a new steroid containing a heterocycle dioxene fused in the A-ring, led to the discovery of a new reaction for which a mechanism is proposed. New structure-activity relationships were established. Some 5ß-steroids, such as compound 4ß,5ß-epoxyandrostan-17-one (9), showed aromatase inhibitory activity, because they adopt a similar A-ring conformation as those of androstenedione, the natural substrate of aromatase. Moreover, new chemical features to increase planarity were disclosed, specifically the 3α,4α-cyclopropane ring, as in 3α,4α-methylen-5α-androstan-17-one (5) (IC50=0.11µM), and the Δ(9-11) double bond in the C-ring, as in androsta-4,9(11)-diene-3,17-dione (13) (IC50=0.25µM). In addition, induced-fit docking (IFD) simulations and site of metabolism (SoM) predictions helped to explain the recognition of new potent steroidal aromatase inhibitors within the enzyme. These insights can be valuable tools for the understanding of the molecular recognition process by the aromatase and for the future design of new steroidal inhibitors.

[Whole-genome sequencing and analysis of a adrostenedione yielding strains Mycobacterium neoaurum MN4].

Objective: Mycobaterium neoaurum MN4 is a substrate-resistant mutant strain with high-yield androstenedione. In order to further study MN4 strain substrate-resistant mechanism and androstenedione biosynthetic pathway, it is necessary to decipher the MN4 strain genome. Methods: The genome was sequenced using highthroughput sequencing technology, and analyzed using relevant software for genome assembly, gene prediction and functional annotation, COG cluster analysis and secondary metabolite biosynthesis gene clusters prediction. Results: The whole genome is assembled into 33 contigs, and the genome size is 5.39 Mb, GC content of 66.9% with encoding 4920 protein genes. The genome sequence was deposited in the GenBank database under the accession number JXYZ00000000. Conclusion: This study is the first report of androstenedione producing strain Mycobacterium neoaurum MN4 genome sequence, and provides a theoretical basis for further heterologous expression of secondary metabolites on Mycobacterium neoaurum MN4.

The isomerization of Δ5-androstene-3,17-dione by the human glutathione transferase A3-3 proceeds via a conjugated heteroannular diene intermediate.

The seemingly simple proton abstraction reactions underpin many chemical transformations, including isomerization reactions, and are thus of immense biological significance. Despite the energetic cost, enzyme-catalyzed proton abstraction reactions show remarkable rate enhancements. The pathways leading to these accelerated rates are numerous and on occasion partly enigmatic. The isomerization of the steroid Δ(5)-androstene-3,17-dione by the glutathione transferase A3-3 in mammals was investigated to gain insight into the mechanism. Particular emphasis was placed on the nature of the transition state, the intermediate suspected of aiding this process, and the hydrogen bonds postulated to be the stabilizing forces of these transient species. The UV-visible detection of the intermediate places this species in the catalytic pathway, whereas fluorescence spectroscopy is used to obtain the binding constant of the analog intermediate, equilenin. Solvent isotope exchange reveals that proton abstraction from the substrate to form the intermediate is rate-limiting. Analysis of the data in terms of the Marcus formalism indicates that the human glutathione transferase A3-3 lowers the intrinsic kinetic barrier by 3 kcal/mol. The results lead to the conclusion that this reaction proceeds through an enforced concerted mechanism in which the barrier to product formation is kinetically insignificant.

QM/MM modeling of the hydroxylation of the androstenedione substrate catalyzed by cytochrome P450 aromatase (CYP19A1).

CYP19A1 aromatase is a member of the Cytochrome P450 family of hemeproteins, and is the enzyme responsible for the final step of the androgens conversion into the corresponding estrogens, via a three-step oxidative process. For this reason, the inhibition of this enzyme plays an important role in the treatment of hormone-dependent breast cancer. The first catalytic subcycle, corresponding to the hydroxilation of androstenedione, has been proposed to occur through a first hydrogen abstraction and a subsequent oxygen rebound step. In present work, we have studied the mechanism of the first catalytic subcycle by means of hybrid quantum mechanics/molecular mechanics methods. The inclusion of the protein flexibility has been achieved by means of Free Energy Perturbation techniques, giving rise to a free energy of activation for the hydrogen abstraction step of 13.5 kcal/mol. The subsequent oxygen rebound step, characterized by a small free energy barrier (1.5 kcal/mol), leads to the hydroxylated products through a highly exergonic reaction. In addition, an analysis of the primary deuterium kinetic isotopic effects, calculated for the hydrogen abstraction step, reveals values (∼10) overpassing the semiclassical limit for the CH, indicating the presence of a substantial tunnel effect. Finally, a decomposition analysis of the interaction energy for the substrate and cofactor in the active site is also discussed. According to our results, the role of the enzymatic environment consists of a transition state stabilization by means of dispersive and polarization effects.

Natural selection acts in opposite ways on correlated hormonal mediators of prenatal maternal effects in a wild bird population.

Maternal hormones are important mediators of prenatal maternal effects. Although many experimental studies have demonstrated their potency in shaping offspring phenotypes, we know remarkably little about their adaptive value. Using long-term data on a wild collared flycatcher (Ficedula albicollis) population, we show that natural selection acts in opposite ways on two maternally derived androgens, yolk androstenedione (A4) and yolk testosterone (T). High yolk A4 concentrations are associated with higher fitness, whereas high yolk T concentrations are associated with lower fitness. Natural selection thus favours females that produce eggs with high A4 and low T concentrations. Importantly, however, there exists a positive (non-genetic) correlation between A4 and T, which suggests that females are limited in their ability to reach this adaptive optimum. Thereby, these results provide strong evidence for an adaptive value of differential maternal androgen deposition, and a mechanistic explanation for the maintenance of variation in maternal investment in the wild.

Resonance Raman spectroscopy of the oxygenated intermediates of human CYP19A1 implicates a compound i intermediate in the final lyase step.

CYP19A1, or aromatase, a cytochrome P450 responsible for estrogen biosynthesis in humans, is an important therapeutic target for the treatment of breast cancer. There is still controversy surrounding the identity of reaction intermediate that catalyzes carbon-carbon scission in this key enzyme. Probing the oxy-complexes of CYP19A1 poised for hydroxylase and lyase chemistries using resonance Raman spectroscopy and drawing a comparison with CYP17A1, we have found no significant difference in the frequencies or isotopic shifts for these two steps in CYP19A1. Our experiments implicate the involvement of Compound I in the terminal lyase step of CYP19A1 catalysis.

Transport of steroid hormones, phytoestrogens, and estrogenic activity across a swine lagoon/sprayfield system.

The inflow, transformation, and attenuation of natural steroid hormones and phytoestrogens and estrogenic activity were assessed across the lagoon/sprayfield system of a prototypical commercial swine sow operation. Free and conjugated steroid hormones (estrogens, androgens, and progesterone) were detected in urine and feces of sows across reproductive stages, with progesterone being the most abundant steroid hormone. Excreta also contained phytoestrogens indicative of a soy-based diet, particularly, daidzein, genistein, and equol. During storage in barn pits and the anaerobic lagoon, conjugated hormones dissipated, and androgens and progesterone were attenuated. Estrone and equol persisted along the waste disposal route. Following application of lagoon slurry to agricultural soils, all analytes exhibited attenuation within 2 days. However, analytes including estrone, androstenedione, progesterone, and equol remained detectable in soil at 2 months postapplication. Estrogenic activity in the yeast estrogen screen and T47D-KBluc in vitro bioassays generally tracked well with analyte concentrations. Estrone was found to be the greatest contributor to estrogenic activity across all sample types. This investigation encompasses the most comprehensive suite of natural hormone and phytoestrogen analytes examined to date across a livestock lagoon/sprayfield and provides global insight into the fate of these analytes in this widely used waste management system.

Isomerization of Δ5-androstene-3,17-dione into Δ4-androstene-3,17-dione catalyzed by human glutathione transferase A3-3: a computational study identifies a dual role for glutathione.

Glutathione transferases (GSTs) are important enzymes in the metabolism of electrophilic xenobiotic and endobiotic toxic compounds. In addition, human GST A3-3 also catalyzes the double bond isomerization of Δ5-androstene-3,17-dione (Δ(5)-AD) and Δ(5)-pregnene-3,20-dione (Δ(5)-PD), which are the immediate precursors of testosterone and progesterone. In fact, GST A3-3 is the most efficient human enzyme known to exist in the catalysis of these reactions. In this work, we have used density functional theory (DFT) calculations to propose a refined mechanism for the isomerization of Δ(5)-AD catalyzed by GST A3-3. In this mechanism the glutathione (GSH) thiol and Tyr9 catalyze the proton transfer from the Δ(5)-AD C4 atom to the Δ(5)-AD C6 atom, with a rate limiting activation energy of 15.8 kcal · mol(-1). GSH has a dual function, because it is also responsible for stabilizing the negative charge that is formed in the O3 atom of the enolate intermediate. The catalytic role of Tyr9 depends on significant conformational rearrangements of its side chain. Neither of these contributions to catalysis has been observed before. Residues Phe10, Leu111, Ala 208, and Ala 216 complete the list of the important catalytic residues. The mechanism detailed here is based on the GST A3-3:GSH:Δ(4)-AD crystal structure and is consistent with all available experimental data.

Cryoradiolysis of oxygenated cytochrome P450 17A1 with lyase substrates generates expected products.

When subjected to γ-irradiation at cryogenic temperatures the oxygenated complexes of Cytochrome P450 CYP17A1 (CYP17A1) bound with either of the lyase substrates, 17α-Hydroxypregnenolone (17-OH PREG) or 17α-Hydroxyprogesterone (17-OH PROG) are shown to generate the corresponding lyase products, dehydroepiandrosterone (DHEA) and androstenedione (AD) respectively. The current study uses gas chromatography-mass spectrometry (GC/MS) to document the presence of the initial substrates and products in extracts of the processed samples. A rapid and efficient method for the simultaneous determination of residual substrate and products by GC/MS is described without derivatization of the products. It is also shown that no lyase products were detected for similarly treated control samples containing no nanodisc associated CYP17 enzyme, demonstrating that the product is formed during the enzymatic reaction and not by GC/MS conditions, nor the conditions produced by the cryoradiolysis process.

Clostridium scindens: a human gut microbe with a high potential to convert glucocorticoids into androgens.

Clostridium scindens American Type Culture Collection 35704 is capable of converting primary bile acids to toxic secondary bile acids, as well as converting glucocorticoids to androgens by side-chain cleavage. The molecular structure of the side-chain cleavage product of cortisol produced by C. scindens was determined to be 11ß-hydroxyandrost-4-ene-3,17-dione (11ß-OHA) by high-resolution mass spectrometry, (1)H and (13)C NMR spectroscopy, and X-ray crystallography. Using RNA-Seq technology, we identified a cortisol-inducible (≈ 1,000-fold) operon (desABCD) encoding at least one enzyme involved in anaerobic side-chain cleavage. The desC gene was cloned, overexpressed, purified, and found to encode a 20α-hydroxysteroid dehydrogenase (HSDH). This operon also encodes a putative "transketolase" (desAB) hypothesized to have steroid-17,20-desmolase/oxidase activity, and a possible corticosteroid transporter (desD). RNA-Seq data suggests that the two-carbon side chain of glucocorticords may feed into the pentose-phosphate pathway and are used as a carbon source. The 20α-HSDH is hypothesized to function as a metabolic "rheostat" controlling rates of side-chain cleavage. Phylogenetic analysis suggests this operon is rare in nature and the desC gene evolved from a gene encoding threonine dehydrogenase. The physiological effect of 11ß-OHAD on the host or other gut microbes is currently unknown.