Hydroxylative activity of Aspergillus niger towards androst-4-ene and androst-5-ene steroids.

Aspergillus niger, one of fungal species most frequently used for experimental and industrial-scale biotransformations of various organic compounds, is generally known to transform steroids at 16ß position. In this work, application of the strain A. niger KCH910 to bioconversion of dehydroepiandrosterone (DHEA), androstenediol and testosterone is described, with emphasis on the metabolic steps leading to the products. Evidence from this study indicated that incubated 5-ene steroids underwent bioconversion within two metabolic pathways: oxidation by the action of 3ß-HSD (3ß-hydroxysteroid dehydrogenase) to 4-ene steroids, and minor allylic hydroxylation to epimeric 7-alcohols. Further transformation of the 3-oxo-4-ene metabolites resulted in non-selective 16-hydroxylation. It is the first report on an A. niger strain able to introduce not only 16ß- but also 16α-hydroxyl function into steroids.

Insight into the orientational versatility of steroid substrates-a docking and molecular dynamics study of a steroid receptor and steroid monooxygenase.

Numerous steroids are essential plant, animal, and human hormones. The medical and industrial applications of these hormones require the identification of new synthetic routes, including biotransformations. The metabolic fate of a steroid can be complicated; it may be transformed into a variety of substituted derivatives. This may be because a steroid molecule can adopt several possible orientations in the binding pocket of a receptor or an enzyme. The present study, based on docking and molecular dynamics, shows that it is indeed possible for a steroid molecule to bind to a receptor binding site in two or more orientations (normal, head-to-tail reversed, upside down). Three steroids were considered: progesterone, dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. Two proteins were employed as hosts: the human mineralocorticoid receptor and a bacterial Baeyer-Villiger monooxygenase. When the steroids were in nonstandard orientations, the estimated binding strength was found to be only moderately diminished and the network of hydrogen bonds between the steroid and the host was preserved.

Biohydroxylation of 7-oxo-DHEA, a natural metabolite of DHEA, resulting in formation of new metabolites of potential pharmaceutical interest.

Metabolism of steroids in healthy and unhealthy human organs is the subject of extensive clinical and biomedical studies. For this kind of investigations, it is essential that the reference samples of new derivatives of natural, physiologically active steroids (especially those difficult to achieve in the chemical synthesis) become available. This study demonstrated for the first time transformation of 7-oxo-DHEA-a natural metabolite of DHEA, using Syncephalastrum racemosum cells. The single-pulse fermentation of substrate produced two new hydroxy metabolites: 1ß,3ß-dihydroxy-androst-5-en-7,17-dione and 3ß,12ß-dihydroxy-androst-5-en-7,17-dione, along with the earlier reported 3ß,9α-dihydroxy-androst-5-en-7,17-dione and 3ß,17ß-dihydroxy-androst-5-en-7-one. Simultaneously, the same metabolites, together with small quantities of 7α- and 7ß-hydroxy-DHEA, as well as the products of their reduction at the C-17 were obtained after transformation of DHEA under pulse-feeding of the substrate. The observed reactions suggested that this micro-organism contains enzymes exhibiting similar activity to those present in human cells. Thus, the resulting compounds can be considered as potential components of the eukaryotic, including human, metabolome.

Microbial Baeyer-Villiger oxidation of 5α-steroids using Beauveria bassiana. A stereochemical requirement for the 11α-hydroxylation and the lactonization pathway.

Beauveria bassiana KCH 1065, as was recently demonstrated, is unusual amongst fungal biocatalysts in that it converts C19 3-oxo-4-ene and 3ß-hydroxy-5-ene as well as 3ß-hydroxy-5α-saturated steroids to 11α-hydroxy ring-D lactones. The Baeyer-Villiger monooxygenase (BVMO) of this strain is distinguished from other enzymes catalyzing BVO of steroidal ketones by the fact that it oxidizes solely substrates with 11α-hydroxyl group. The current study using a series of 5α-saturated steroids (androsterone, 3α-androstanediol and androstanedione) has highlighted that a small change of the steroid structure can result in significant differences of the metabolic fate. It was found that the 3α-stereochemistry of hydroxyl group restricted "normal" binding orientation of the substrate within 11α-hydroxylase and, as a result, androsterone and 3α-androstanediol were converted into a mixture of 7ß-, 11α- and 7α-hydroxy derivatives. Hydroxylation of androstanedione occurred only at the 11α-position, indicating that the 3-oxo group limits the alternative binding orientation of the substrate within the hydroxylase. Only androstanedione and 3α-androstanediol were metabolized to hydroxylactones. The study uniquely demonstrated preference for oxidation of equatorial (11α-, 7ß-) hydroxyketones by BVMO from B. bassiana. The time course experiments suggested that the activity of 17ß-HSD is a factor determining the amount of produced ring-D lactones. The obtained 11α-hydroxylactones underwent further transformations (oxy-red reactions) at C-3. During conversion of androstanedione, a minor dehydrogenation pathway was observed with generation of 11α,17ß-dihydroxy-5α-androst-1-en-3-one. The introduction of C1C2 double bond has been recorded in B. bassiana for the first time.

Hydroxylation of DHEA and its analogues by Absidia coerulea AM93. Can an inducible microbial hydroxylase catalyze 7α- and 7ß-hydroxylation of 5-ene and 5α-dihydro C19-steroids?

In this paper we focus on the course of 7-hydroxylation of DHEA, androstenediol, epiandrosterone, and 5α-androstan-3,17-dione by Absidia coerulea AM93. Apart from that, we present a tentative analysis of the hydroxylation of steroids in A. coerulea AM93. DHEA and androstenediol were transformed to the mixture of allyl 7-hydroxy derivatives, while EpiA and 5α-androstan-3,17-dione were converted mainly to 7α- and 7ß-alcohols accompanied by 9α- and 11α-hydroxy derivatives. On the basis of (i) time course analysis of hydroxylation of the abovementioned substrates, (ii) biotransformation with resting cells at different pH, (iii) enzyme inhibition analysis together with (iv) geometrical relationship between the C-H bond of the substrate undergoing hydroxylation and the cofactor-bound activated oxygen atom, it is postulated that the same enzyme can catalyze the oxidation of C7-Hα as well as C7-Hß bonds in 5-ene and 5α-dihydro C19-steroids. Correlations observed between the structure of the substrate and the regioselectivity of hydroxylation suggest that 7ß-hydroxylation may occur in the normal binding enzyme-substrate complex, while 7α-hydroxylation-in the reverse inverted binding complex.

Biotransformations utilizing ß-oxidation cycle reactions in the synthesis of natural compounds and medicines.

ß-Oxidation cycle reactions, which are key stages in the metabolism of fatty acids in eucaryotic cells and in processes with a significant role in the degradation of acids used by microbes as a carbon source, have also found application in biotransformations. One of the major advantages of biotransformations based on the ß-oxidation cycle is the possibility to transform a substrate in a series of reactions catalyzed by a number of enzymes. It allows the use of sterols as a substrate base in the production of natural steroid compounds and their analogues. This route also leads to biologically active compounds of therapeutic significance. Transformations of natural substrates via ß-oxidation are the core part of the synthetic routes of natural flavors used as food additives. Stereoselectivity of the enzymes catalyzing the stages of dehydrogenation and addition of a water molecule to the double bond also finds application in the synthesis of chiral biologically active compounds, including medicines. Recent advances in genetic, metabolic engineering, methods for the enhancement of bioprocess productivity and the selectivity of target reactions are also described.