Hydroxylation Of Dehydroepiandrosterone Research Articles

Identification of a fungal cytochrome P450 with steroid two-step ordered selective hydroxylation characteristics in Colletotrichum lini

Microbial hydroxylation reaction has greatly enriched the number of steroids and created many meaningful new compounds. The dihydroxylation of dehydroepiandrosterone (DHEA) by filamentous fungi produces an important product 3β,7α,15α-trihydroxy-5-androstene-17-one (7α,15α-diOH-DHEA), which can be used as a key intermediate for the synthesis of contraceptive drospirenone. The introduction of microbial hydroxylation reaction reduces the traditional chemical synthesis process by 4 steps and greatly improves the productivity and economic efficiency. Colletotrichum lini is an industrial strain producing 7α,15α-diOH-DHEA, but the related cytochrome P450 that plays hydroxylation effect has not yet been discovered. In this work, a combination of quantitative proteomics, qRT-PCR, and functional expression in Pichia pastoris was used to identify highly induced steroid hydroxylase from Colletotrichum lini ST-1. A novel fungal cytochrome P450 monooxygenase CYP68JX was identified. The biotransformation in recombinant yeast confirmed that the cytochrome P450 has steroid C7α and C15α hydroxylase activities. The hydroxylation of DHEA by CYP68JX is an ordered reaction, proceeding from the C7 to the C15 site of the steroidal nucleus. The cloning and identification of the CYP68JX gene provide useful information for deepening the understanding regarding the structural basis of its regional and stereoselectivity.

Production of 7α,15α-diOH-DHEA from dehydroepiandrosterone by Colletotrichum lini ST-1 through integrating glucose-feeding with multi-step substrate addition strategy.

Hydroxylation of dehydroepiandrosterone (DHEA) to 3β,7α,15α-trihydroxy-5-androstene-17-one (7α,15α-diOH-DHEA) by Colletotrichum lini ST-1 is an essential step in the synthesis of many steroidal drugs, while low DHEA concentration and 7α,15α-diOH-DHEA production are tough problems to be solved urgently in industry. In this study, the significant improvement of 7α,15α-diOH-DHEA yield in 5-L stirred fermenter with 15g/L DHEA was achieved. To maintain a sufficient quantity of glucose for the bioconversion, glucose of 15g/L was fed at 18h, the 7α,15α-diOH-DHEA yield and dry cell weight were increased by 17.7 and 30.9%, respectively. Moreover, multi-step DHEA addition strategy was established to diminish DHEA toxicity to C. lini, and the 7α,15α-diOH-DHEA yield raised to 53.0%. Further, a novel strategy integrating glucose-feeding with multi-step addition of DHEA was carried out and the product yield increased to 66.6%, which was the highest reported 7α,15α-diOH-DHEA production in 5-L stirred fermenter. Meanwhile, the conversion course was shortened to 44h. This strategy would provide a possible way in enhancing the 7α,15α-diOH-DHEA yield in pharmaceutical industry.

Effects on DHEA levels by estrogen in rat astrocytes and CNS co-cultures via the regulation of CYP7B1-mediated metabolism

The neurosteroid dehydroepiandrosterone (DHEA) is formed locally in the CNS and has been implicated in several processes essential for CNS function, including control of neuronal survival. An important metabolic pathway for DHEA in the CNS involves the steroid hydroxylase CYP7B1. In previous studies, CYP7B1 was identified as a target for estrogen regulation in cells of kidney and liver. In the current study, we examined effects of estrogens on CYP7B1-mediated metabolism of DHEA in primary cultures of rat astrocytes and co-cultures of rat CNS cells. Astrocytes, which interact with neurons in several ways, are important for brain neurosteroidogenesis. We found that estradiol significantly suppressed CYP7B1-mediated DHEA hydroxylation in primary mixed CNS cultures from fetal and newborn rats. Also, CYP7B1-mediated DHEA hydroxylation and CYP7B1 mRNA were markedly suppressed by estrogen in primary cultures of rat astrocytes. Interestingly, diarylpropionitrile, a well-known agonist of estrogen receptor β, also suppressed CYP7B1-mediated hydroxylation of DHEA. Several previous studies have reported neuroprotective effects of estrogens. The current data indicate that one of the mechanisms whereby estrogen can exert protective effects in the CNS may involve increase of the levels of DHEA by suppression of its metabolism.

Microbial hydroxylation of dehydroepiandrosterone

Microbial hydroxylation of dehydroepiandrosterone

Screening of mycelial fungi for 7α- and 7β-hydroxylase activity towards dehydroepiandrosterone

In total, 481 fungal strains were screened for the ability to carry out 7(α/β)-hydroxylation of dehydroepiandrosterone (DHEA, 3β-hydroxy-5-androsten-17-one). Representatives of 31 genera of 15 families and nine orders of ascomycetes, 17 genera of nine families and two orders of zygomycetes, two genera of two families and two orders of basidiomycetes, and 14 genera of mitosporic fungi expressed 7(α/β)-hydroxylase activity. The majority of strains were able to introduce a hydroxyl group to position 7α. Active strains selectively producing 3β,7α-dihydroxy-5-androsten-17-one were found among Actinomucor, Backusella, Benjaminiella, Epicoccum, Fusarium, Phycomyces and Trichothecium, with the highest yield of 1.25 and 1.9 g L−1 from 2 and 5 g L−1 DHEA, respectively, reached with F. oxysporum. Representatives of Acremonium, Bipolaris, Conidiobolus and Curvularia formed 3β,7β-dihydroxy-5-androsten-17-one as a major product from DHEA. The structures of the major steroid products were confirmed by TLC, gas chromatography (GC), mass spectra (MS), and 1H-NMR analyses.

Ergosteroids V: preparation and biological activity of various D-ring derivatives in the 7-oxo-dehydroepiandrosterone series.

Our previous finding that D-ring seco derivatives of dehydroepiandrosterone retained biologic activity (Reich et al., Steroids 1998;63:542-53) motivated us to synthesize and test a number of steroids in which the D-ring is retained but altered in various ways. Several new steroids were synthesized and characterized by (1)H and (13)C NMR spectroscopy. The availability of a number of closely related compounds allowed detailed (13)C chemical shift correlations. Using the induction of two thermogenic enzymes in rats, liver mitochondrial glycerophosphate dehydrogenase (GPDH) and cytosolic malic enzyme, as criteria of biologic activity some 30 compounds were assayed. Hydroxylation of dehydroepiandrosterone (DHEA) at the 16 alpha position was previously shown to diminish activity (Lardy et al., Steroids 1998;63:158-65); the corresponding 7-oxo compound is fully active. Hydroxylation at the 15 beta position of DHEA, 7-oxo-DHEA, or 16 alpha-hydroxy-7-oxo-DHEA greatly diminished the induction of GPDH but induction of malic enzyme was retained. Most 5,15 diene steroids tested had 2 weak, or no, ability to enhance the formation of GPDH but did increase malic enzyme.

Neurosteroid metabolism. 7 alpha-Hydroxylation of dehydroepiandrosterone and pregnenolone by rat brain microsomes.

Two 'neurosteroids', dehydroepiandrosterone (DHEA) and pregnenolone (PREG), are converted by rat brain microsomes into polar metabolites, identified as the respective 7 alpha-hydroxylated (7 alpha-OH) derivatives by the 'twin ion' technique of g.l.c.-m.s. with deuterated substrates. The enzymic reaction requires NADPH and is stimulated 2-4-fold by EDTA. Under optimal conditions (pH 7.4, 0.5 mM-NADPH, 1 mM-EDTA), the Km values for DHEA and PREG are 13.8 and 4.4 microM respectively, and the Vmax. values are 322 and 38.8 pmol/min per mg of microsomal protein respectively. Trace amounts of putative 7 beta-OH derivatives of DHEA and PREG are detected. Oestradiol, at a pharmacological concentration of 5 microM, inhibits DHEA and PREG 7 alpha-hydroxylation. Formation of 7 alpha-hydroxylated metabolites is low in prepubertal rats and increases 5-fold in adults. Derivatives of PREG and DHEA, such as PREG sulphate, DHEA sulphate, progesterone and 3 alpha-hydroxy-5 alpha-pregnan-20-one, are known to be neuroactive. Therefore the quantitatively important metabolism to 7 alpha-OH compounds may contribute to the control of neurosteroid activity in brain.

Hydroxylation of dehydroepiandrosterone in human scalp hair follicles.

The dehydroepiandrosterone hydroxylating enzyme system in the hair follicle has been characterized. It is found in the particulate fraction of the follicle, and is inhibited by carbon monoxide and the cytochrome P450 inhibitor metyrapone, and appears to be dependent upon NADPH. In these major characteristics the hair follicle enzyme is very similar to the liver monooxygenase system.

Hydroxylation of dehydroepiandrosterone in the eye lens

Hydroxylation of the steroid hormone dehydroepiandrosterone in the calf lens is inhibited by carbon monoxide and stimulated by NADPH. The enzyme concerned was found to be membrane-bound. Although the enzyme resembles the liver mono-oxygenase system in these characteristics, the presence of cytochrome P-450 in the lens could not be proved by measuring a difference spectrum with carbon monoxide, probably because the concentration of the enzyme is too low. Preparations of purified lens fiber plasma membranes also hydroxylate dehydroepiandrosterone. This indicates that the fiber plasma membranes act as supports for enzyme complexes. In this respect they resemble cytoplasmic membranes and plasma membranes derived from other tissues. Cultured lens cell contain the hydroxylating enzyme, although its activity is dependent on the culture conditions used. It is striking that in lens fibers the enzyme which seems to convert dehydroepiandrosterone specifically occurs on the plasma membranes, whereas, for instance, in liver, hemoproteins localized on the endoplasmic reticulum, exert hydroxylation activity towards a variety of steroids. This suggests some regulatory role for dehydroepiandrosterone in lens growth and metabolism.

7 -Hydroxylation of dehydroepiandrosterone in human testis and epididymis in vitro.

Zerhackte menschliche Hoden und Epididymis wurden mit Dehydroepiandrosteron ohne Kofaktoren bebrutet und es wurden unter anderem 7α-und 7β-Hydroxydehydroepiandrosteron und 5-Androsten-3β, 7α, 17β-triol charakterisiert. Die Ausbeute an 7-hydroxylierten Metaboliten war fast gleich in Hoden und Epididymis.

Hydroxylation of dehydroepiandrosterone and its acetate with potato tuber slices

Evidence is presented for the view that the potato tuber tissue contains an unstable enzyme capable of hydroxylation of dehydroepiandrosterone and its acetate in low yield. The products of hydroxylation were isolated and identified. The possibility is discussed that auto-oxidation is a simultaneous reaction.