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.

Investigating the biosynthesis and metabolism of 11ß-hydroxyandrostenedione.

The "rediscovery" 11ß-hydroxyandrostenedione (11OHA4) placed the spotlight on this unique adrenal-derived hormone with researchers and clinicians once again focusing on the steroid's presence in endocrine pathology. Little was known about the steroid other than its chemical characterisation and that a mitochondrial cytochrome P450 enzyme catalysed the 11ß-hydroxylation of 11OHA4. The fact that neither the biosynthesis nor metabolism of 11OHA4 had been fully characterised presented an ideal opportunity to investigate the metabolic pathways. In addition, methodologies and analytical tools have improved vastly since 11OHA4 was first identified in the 1950s. Cell models, recombinant DNA technology and steroid quantification using liquid chromatography mass spectrometry have greatly facilitated investigations in the field of steroidogenesis. Evident from the structure is that 11OHA4 can be metabolised by hydroxysteroid dehydrogenases and reductases acting on the C4/C5 double bond and on functional moieties at specific carbons on the cyclopentane-perhydro-phenanthrene backbone of the steroid. In this chapter, the biosynthesis and metabolism of 11OHA4 is followed using two strategies that complement each another; (i) human cell models either transiently transfected with recombinant DNA or expressing endogenous steroidogenic enzymes and (ii) steroid identification and quantification using high resolution mass spectrometry. These methodologies have proven invaluable in the determination of 11OHA4's metabolic route. Both strategies are presented with the focus on the accurate identification and quantification of steroids using UHPLC-MS/MS and UPC2-MS/MS. The protocols described in this chapter lay a sound foundation which can aid the researcher and be adapted and implement in future studies.

Postpartum nutrition affects the insulin-like growth factor system in dominant follicles and plasma of anestrous beef cows.

Effects of nutrition on insulin-like growth factor-I (IGF-I), IGF binding proteins (IGFBP), and insulin in plasma and dominant follicles were evaluated at day 72 and 56 (Exp. 1, n = 12 and Exp. 2, n = 28, respectively) postpartum in anovulatory primiparous beef cows. Cows were stratified based on body condition score at calving and randomly assigned to nutritional treatments: maintain (M), 2.27 kg of a 40 % CP supplement per day and ad libitum hay; or gain (G), ad libitum access to a 50 % concentrate diet and ad libitum hay. Blood samples were collected twice weekly starting 30 days postpartum. Ovarian follicles were evaluated using ultrasonography commencing 42 (Exp. 1) or 30 (Exp. 2) days postpartum. Body weight and condition score were greater (P < 0.05) for cows of G than M groups and postpartum interval to luteal function was longer for cows of the M than G group. Insulin and IGF-I concentrations in follicular fluid (FF) and plasma were greater (P < 0.05) for cows of the G than M group at follicular aspiration. Plasma and FF IGFBP4 and IGFBP5 concentrations were greater (P <  0.05) in Exp. 2, and IGFBP5 was greater in Exp. 1 for cows of the G than M group. Treatment did not affect FF steroid concentrations or granulosal cell CYP19A1, PAPPA, IGFBP4, and IGFBP5 mRNA abundance. These results indicate concentrations of IGF-I, insulin, IGFBP4, and IGFBP5 in FF and plasma are affected by nutritional intake and may be related to follicular function.

Innovative C2-symmetric testosterone and androstenedione dimers: Design, synthesis, biological evaluation on prostate cancer cell lines and binding study to recombinant CYP3A4.

The synthesis of two isomeric testosterone dimers and an androstenedione dimer is reported. The design takes advantage of an efficient transformation of testosterone leading to the synthesis of the key diene, 7α-(buta-1,3-dienyl)-4-androsten-17ß-ol-3-one, through an elimination reaction. It was found that in some instances the same reaction led to partial epimerization of the 17ß-hydroxyl group into the 17α-hydroxyl group. The specific orientation of the hydroxyl function was confirmed by NMR spectroscopy. Capitalizing on this unforeseen side reaction, several dimers were assembled using an olefin metathesis reaction with Hoveyda-Grubbs catalyst. This led to the formation of two isomeric testosterone dimers with 17α-OH or 17ß-OH (14α and 14ß) as well as an androstenedione dimer (14). The new dimers and their respective precursors were tested on androgen-dependent (LNCaP) and androgen independent (PC3 and DU145) prostate cancer cells. It was discovered that the most active dimer was made of the natural hormone testosterone (14ß) with an average IC50 of 13.3 µM. In LNCaP cells, 14ß was ∼5 times more active than the antiandrogen drug cyproterone acetate (IC50 of 12.0 µM vs. 59.6 µM, respectively). At low concentrations (0.25-0.5 µM), 14α and 14ß were able to completely inhibit LNCaP cell growth induced by testosterone or dihydrotestosterone. Furthermore, cross-reactivity of androgen-based dimers with sterol-metabolizing cytochrome P450 3A4 was explored and the results are disclosed herein.

Back where it belongs: 11ß-hydroxyandrostenedione compels the re-assessment of C11-oxy androgens in steroidogenesis.

Adrenal steroidogenesis has, for decades, been depicted as three biosynthesis pathways -the mineralocorticoid, glucocorticoid and androgen pathways with aldosterone, cortisol and androstenedione as the respective end products. 11ß-hydroxyandrostenedione was not included as an adrenal steroid despite the adrenal output of this steroid being twice that of androstenedione. While it is the end of the line for aldosterone and cortisol, as it is in these forms that they exhibit their most potent receptor activities prior to inactivation and conjugation, 11ß-hydroxyandrostenedione is another matter entirely. The steroid, which is weakly androgenic, has its own designated pathway yielding 11-ketoandrostenedione, 11ß-hydroxytestosterone and the potent androgens, 11-ketotestosterone and 11-ketodihydrotestosterone, primarily in the periphery. Over the last decade, these C11-oxy C19 steroids have once again come to the fore with the rising number of studies contradicting the generally accepted notion that testosterone and it's 5α-reduced product, dihydrotestosterone, are the principal potent androgens in humans. These C11-oxy androgens have been shown to contribute to the androgen milieu in adrenal disorders associated with androgen excess and in androgen dependant disease progression. In this review, we will highlight these overlooked C11-oxy C19 steroids as well as the C11-oxy C21 steroids and their contribution to congenital adrenal hyperplasia, polycystic ovarian syndrome and prostate cancer. The focus is on new findings over the past decade which are slowly but surely reshaping our current outlook on human sex steroid biology.

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.

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.

Fungal transformation of norandrostenedione with Cunninghamella blakesleeana and anti-bacterial activity of the transformed products.

Although the discovery of antibiotics has decreased the spread and severity of infectious diseases, their uncontrolled use has lead to the emergence of bacterial resistance to existing chemotherapeutic agents. Bacterial disease thus remains a challenge for health authorities in worldwide and especially in sub-Saharan Africa. Despite their efficacy, the miss-use of medicinal plants for the treatment of infectious diseases couple to the farming and hunting activities has contribute enormously to the destruction of many medicinal plant species. In search of an alternative for new and effective agents against bacterial infection, norandrostenedion (19-nor-4-androsten-3,17-dione) (1), was biotransformed by Cunninghamella blakesleeana ATCC 8688A and yielded a new metabolite, 6α,10 ß -dihydroxy-19-nor-4-androsten-3-one (2), together with three known compounds, 10 ß -hydroxy-19-nor-4-androsten-3,17-dione (3), 6 ß,10 ß,17 ß -trihydroxy-19-nor-4-androsten-3-one (4) and 10 ß,17 ß -dihydroxy-19-nor-4-androsten-3-one (5). Their structures were elucidated on the basis ofspectroscopic techniques: NMR analysis (1D and 2D) and HRIE-MS and by comparison with previously reported data. In addition, the agar diffusion method was used to evaluate the diameter of the inhibition zone and INT colorimetric assay for MIC values. All metabolites obtained showed a potent and varied activity against tested bacteria. These results support the uses of biotransformation to develop new antimicrobial compounds for clinical application.

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.

An Efficient Procedure for the Synthesis of 21-Acetoxypregna-1,4,9(11),16- tetraene-3,20-dione.

BACKGROUND: Halogenated corticosteroids are widely used in medicine, and the global need of these steroidal APIs is estimated to be 40 - 70 tons, annually. Vietnam currently imports the pharmaceutical compounds up to 90%, in particular 100% of steroidal drugs. Currently, industrial production is based on the chemical syntheses of corticosteroids from either 16- dehydropregnenolone acetate (obtained from diosgenin) or androstenedione (obtained from phytosterol). The development of shorter synthetic schemes and more economically feasible technologies is of great significance. Introduction of 1(2)-double bond at the final stages of the corticosteroids synthesis results inpoor yield. 21-Acetoxypregna-1,4,9(11),16-tetraene-3,20-dione (tetraene acetate) is a key intermediate in the synthesis of highly active halogenated corticosteroids such as dexamethasone and other halogenated corticosteroids. 21-acetoxypregna-1,4,9(11),16- tetraene-3,20-dione is a key intermediate in the synthesis of dexamethasone from the readily available and cheap 9α-hydroxyandrost-4-ene-3,17-dione. OBJECTIVE: The purpose of this study was the development of an efficient and shorter procedure for the synthesis of 21-acetoxypregna-1,4,9(11),16-tetraene-3,20-dione from 9α-hydroxyandrostenedione, which is a product of a bio-oxidative degradation of the side chain of phytosterols. METHODS: Pregnane side chain was constructed using cyanohydrin method. For 1(2)- dehydrogenation, selene dioxide was applied for the introduction of Δ1(2)-double bond. Other stages of the synthesis were epimerization, Stork's iodination procedure and dehydration. RESULT: 21-Acetoxypregna-1,4,9(11),16-tetraene-3,20-dione was prepared from 9α- hydroxyandrostenedione in yield more than 46%. CONCLUSION: An efficient and practically feasible procedure for the synthesis of 21-acetoxypregna- 1,4,9(11),16-tetraene-3,20-dione from 9α-hydroxyandrostenedione, a key intermediate for the synthesis of 9-haloidated corticoids, has been developed. The procedure can be applied for the production of value-added 9-haloidated corticoids.

The 11ß-hydroxyandrostenedione pathway and C11-oxy C21 backdoor pathway are active in benign prostatic hyperplasia yielding 11keto-testosterone and 11keto-progesterone.

In clinical approaches to benign prostatic hyperplasia (BPH) and prostate cancer (PCa), steroidogenesis or the disruption thereof is the main thrust in treatments restricting active androgen production. Extensive studies have been undertaken focusing on testosterone and dihydrotestosterone (DHT). However, the adrenal C11-oxy C19 steroid, 11ß-hydroxyandrostenedione (11OHA4), also contributes to the active androgen pool in the prostate microenvironment, and while it has been shown to impact castration resistant prostate cancer, the C11-oxy C19 steroids together with the C11-oxy C21 steroids have not been studied in BPH. The study firstly investigated the metabolism of these adrenal steroids in the BPH-1 model. Comprehensive profiles identified 11keto-testosterone as the predominant active androgen in the metabolism of the C11-oxy C19 steroids, and we identified, for the first time, 11ß-hydroxy-5α-androstane-3α,17ß-diol, a novel steroid in the 11OHA4-pathway. Analysis of the inactivation and reactivation of the metabolites showed that DHT is more readily inactivated than 11keto-dihydrotestosterone (11KDHT). The conversion of 11ß-hydroxyprogesterone (11ßOHPROG) yielded 11keto-progesterone (11KPROG), while the latter yielded 11keto-dihydroprogesterone (11KDHPROG). BPH tissue analysis identified high levels of 11ß-hydroxyandrosterone (4-14 ng/g) and 11keto-androsterone (9-160 ng/g), together with androstenedione (A4; ∼7.5 ng/g). The major C11-oxy C21 steroids detected were 11ßOHPROG (∼46 ng/g), 11KPROG (∼130 ng/g) as well as 11KDHPROG (∼282 ng/g). While circulatory 11ßOHPROG was detected below the limit of quantification, 11KPROG and 11KDHPROG were detected at 6 and 8.5 nmol/L, respectively. Glucuronide derivatives of both 11KPROG and pregnanetriol were also detected. 11OHA4 was the major free androgen in circulation at 85.9 nmol/L, ±12-fold higher than A4, together with 5α-androstane-3α,17ß-diol quantified at 69.3 nmol/L. Circulatory C11-oxy C19 steroids levels were also significantly higher (8-fold) than the C11-oxy C21 steroid levels, while the former were similar to the C19 steroid levels, in contrast to levels in PCa. The study highlights the contribution of adrenal C11-oxy steroids to the androgen pool in BPH underscoring their limited reactivation and elimination, and significant inter-individual variations regarding steroid levels and conjugation. Targeted steroid metabolome analysis is critical to understanding prostate steroidogenesis and disease progression, and analysis of circulatory C11-oxy C19 and C11-oxy C21 steroids, together with intraprostatic levels, add to our current understanding of BPH.

Biotransformation of androstenedione and androstadienedione by selected Ascomycota and Zygomycota fungal strains.

Filamentous fungi is a huge phylum of lower eukaryotes with diverse activities towards various substrates, however, their biocatalytic potential towards steroids remains greatly underestimated. In this study, more than forty Ascomycota and Zygomycota fungal strains of 23 different genera were screened for the ability to catalyze structural modifications of 3-oxo-androstane steroids, - androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD). Previously unexplored for these purposes strains of Absidia, Acremonium, Beauveria, Cunninghamella, Doratomyces, Drechslera, Fusarium, Gibberella genera were revealed capable of producing in a good yield valuable 7α-, 7ß-, 11α- and 14α-hydroxylated derivatives, as well as 17ß-reduced and 1(2)-dehydrogenated androstanes. The bioconversion routes of AD and ADD were proposed based on the key intermediates identification and time courses of the bioprocesses. Six ascomycete strains were discovered to provide effective 7ß-hydroxylation of ADD which has not been so far reported. The structures of major products and intermediates were confirmed by HPLC, mass-spectrometry (MS), 1H and 13C NMR analyses. The results contribute to the knowledge on the functional diversity of steroid-transforming filamentous fungi. Previously unexplored fungal biocatalysts capable of effective performing structural modification of AD and ADD can be applied for industrial bioprocesses of new generation.

A biocatalytic hydroxylation-enabled unified approach to C19-hydroxylated steroids.

Steroidal C19-hydroxylation is pivotal to the synthesis of naturally occurring bioactive C19-OH steroids and 19-norsteroidal pharmaceuticals. However, realizing this transformation is proved to be challenging through either chemical or biological synthesis. Herein, we report a highly efficient method to synthesize 19-OH-cortexolone in 80% efficiency at the multi-gram scale. The obtained C19-OH-cortexolone can be readily transformed to various synthetically useful intermediates including the industrially valuable 19-OH-androstenedione, which can serve as a basis for synthesis of C19-functionalized steroids as well as 19-nor steroidal drugs. Using this biocatalytic C19-hydroxylation method, the unified synthesis of six C19-hydroxylated pregnanes is achieved in just 4 to 9 steps. In addition, the structure of sclerosteroid B is revised on the basis of our synthesis.

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.

Cross-method comparison of serum androstenedione measurement with respect to the validation of a new fully automated chemiluminescence immunoassay.

OBJECTIVES: Androstenedione is an androgen produced as an intermediate product of the biosynthesis of testosterone and estradiol in testicles, ovaries and also in the adrenal cortex. Measurement is used for diagnosing and differentiating hirsutism and virilisation, enzyme deficiencies of the steroid hormone biosynthesis, and in suspicion of androgen-producing tumors. METHODS: Specimens included de-identified residual serum specimens submitted for routine testing and banked adult and pediatric sera. Samples were measured with tandem mass spectrometry, two automated immunoassays, the newly developed DiaSorin LIAISON androstenedione assay, the Immulite assay, and a radioimmunoassay (Beckman Coulter) according to manufacturer's protocols. All methods were correlated, and the analytical sensitivity, linearity and imprecision of each assay determined. Diagnostic accuracy with respect to detection of PCOS in women was evaluated by verifying the respective reference ranges of the different assays and receiver operating characteristic (ROC) curve analysis. RESULTS: Due to the methodology, LC-MS/MS demonstrated the highest analytical specificity, good performance and excellent diagnostic accuracy. The best agreement was found with the LIAISON chemiluminescent immunoassay method. Due to its lower analytical sensitivity, the measured values in children were often outside the measuring range. Although, the coefficient of correlation between LC-MS/MS and the Beckman Coulter radioimmunoassay was lower, the assay demonstrated the best analytical sensitivity and a similar diagnostic accuracy in adults. The Immulite androstenedione chemiluminescent immunoassay showed the poorest performance and was not interchangeable with the other assays. CONCLUSIONS: These data suggest the LIAISON androstenedione assay may be a suitable alternative for the measurement of androstenedione in serum of adult patients with all advantages of a fully automated assay.

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.

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.

Androgen- and estrogen-receptor mediated activities of 4-hydroxytestosterone, 4-hydroxyandrostenedione and their human metabolites in yeast based assays.

4-Hydroxyandrost-4-ene-3,17-dione, also named formestane, is an irreversible aromatase inhibitor and therapeutically used as anti-breast cancer medication in post-menopausal women. Currently, no therapeutical indication led to approval of its 17-hydroxylated analog 4-hydroxytestosterone, an anabolic steroid. However, it is currently investigated in a clinical trial for breast cancer. In context with sports doping, aromatase inhibitors are administered to reduce estrogenic side effects of misused anabolic substances or their metabolites. Therefore, both substances are prohibited in sports by the World Anti-Doping Agency (WADA). Analysis of urinary phase I and phase II metabolites showed similar results for both compounds. In the current investigation, 4-hydroxyandrost-4-ene-3,17-dione, 4-hydroxytestosterone and seven of their described urinary metabolites as well as 2α-hydroxyandrostenedione were tested in the yeast androgen screen and the yeast estrogen screen. Androgenic effects were observed for all tested substances, except for one, which showed anti-androgenic properties. With regard to the yeast estrogen screen, estrogenic effects were observed for only two metabolites at rather high concentrations, while six out of the ten substances tested showed anti-estrogenic properties. In terms of the strong androgenic effect observed for 4-hydroxytestosterone (10-8 M), 4-hydroxyandrost-4-ene-3,17-dione (10-8 M) and two more urinary metabolites, the yeast androgen assay may also be used to trace abuse in urine samples.

Refining androstenedione and bisnorcholenaldehyde from mother liquor of phytosterol fermentation using macroporous resin column chromatography followed by crystallization.

Androstenedione is an androgen and intermediate in the biosynthesis of most adrenocortical, anabolic, sex and synthetic steroids, such as canrenone, eplerenone, norethindrone and spironolactone. Bisnorcholenaldehyde is an important intermediate in the synthesis of progesterone. This study established an androstenedione and bisnorcholenaldehyde separation method that used a macroporous adsorption resin and an ethanol-water mixture as eluent. The adsorption properties of 12 non-polar or weakly polar macroporous adsorption resins were compared, and three resins exhibited a high adsorption capacity and high desorption rate for both androstenedione and bisnorcholenaldehyde. The three resins were then compared using column chromatography, and one resin was selected and parameters (flow rate, resin size, ethanol concentration and volume) of chromatography were optimized to obtain high purity and recovery. Chromatography eluate was concentrated, dissolved in suitable solvent and crystallized at an optimal temperature to obtain a high purity of both androstenedione and bisnorcholenaldehyde from the same starting material. The levels of androstenedione and bisnorcholenaldehyde in the raw material were 39.78% and 19.15%, respectively. After preparative separation and enrichment by resin column chromatography and crystallization, the purity of androstenedione and bisnorcholenaldehyde was 94.3% and 98.6%, respectively, with their recovery yields of 66.8% and 57.9%, respectively. In addition, the resin maintained over 90% separation efficiency for 5 cycles of adsorption. These results indicated that the combination of macroporous resin chromatography followed by crystallization provide a simple, effective, environmentally friendly and low-cost method for the simultaneous purification of androstenedione and bisnorcholenaldehyde.

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.