Vera M. Nikolayeva

Extracellular 3β-hydroxysteroid oxidase of Mycobacterium vaccae VKM Ac-1815D

Extracellular 3beta-hydroxysteroid oxidase (SO) has been isolated from cell-free cultivation broth at the growth of Mycobacterium vaccae VKM Ac-1815D on glycerol-mineral medium in the presence of sitosterol. The enzyme is responsible for the transformation of 3beta-hydroxy-5-ene- to 3-keto-4-ene-moiety of steroids including dehydrogenation of 3beta-hydroxy function followed by delta5-->delta4 isomerization. 6-Hydroxy-4-sitosten-3-one and 6-hydroxy-4-androsten-3,17-dione were revealed among the metabolites at the incubation of the enzyme preparations with sitosterol and dehydroepiandrosterone (DHEA), respectively. The enzyme was strongly NADH or NADPH dependent. SO has been purified over 300-fold using cultivation broth concentration on hollow fibers followed by fractionation by ammonium sulphate, column chromatography on DEAE-Toyopearl, hydroxyapatite Bio-Gel HTP and double gel-filtration on Bio-Gel A 0.5 M. SDS-electrophoresis gave a molecular mass estimate of 62 +/- 4 kDa. The purified SO obeyed Michaelis-Menten kinetics, double reciprocal plots kinetics revealed Km value towards DHEA 5 x 10(-4) M. Along with SO activity, 17-hydroxysteroid dehydrogenase (17-OH SDH) and 3-ketosteroid-1(2)-dehydrogenase (1(2)-SDH) activities were detected in cell-free cultivation broth. The extracellular steroid transforming activities of C-17-ketosteroid producing mycobacteria were hitherto unreported.

Cholesterol oxidase ChoD is not a critical enzyme accounting for oxidation of sterols to 3-keto-4-ene steroids in fast-growing Mycobacterium sp. VKM Ac-1815D

Fast-growing strain of Mycobacterium sp. VKM Ac-1815D is capable of effective oxidizing of sterols (phytosterol, cholesterol, ergosterol) to androstenedione and other valuable 3-oxo-steroids. To elucidate the role of cholesterol oxidase in sterol catabolism by the strain, the choD gene has been cloned and sequenced. The deduced gene product (M(r) 63.5kDa) showed homologies over its entire length to a large number of proteins belonging to the InterPro-family EPR006076, which includes various FAD dependent oxidoreductases. The expression of choD in Escherichia coli was shown to result in the synthesis of membrane associated cholesterol oxidase. In addition to cholesterol, the enzyme oxidized β-sitosterol, dehydroepiandrosterone, ergosterol, pregnenolone, and lithocholic acid. Knock-out of choD in Mycobacterium sp. VKM Ac-1815D strain was obtained by the gene replacement technique. The mutant strain transformed sitosterol forming exclusively 3-keto-4-ene steroids with androstenedione as a major product, thus evidencing that choD knock out did not abrogate sterol A-ring oxidation. The results indicated that ChoD is not a critical enzyme responsible for modification of 3β-hydroxy-5-ene- to 3-keto-4-ene steroids in Mycobacterium sp. VKM Ac-1815D. Article from a special issue on steroids and microorganisms.

17-Hydroxysteroid dehydrogenases of Mycobacterium sp. VKM Ac-1815D mutant strain

Whole cells and crude extract of Mycobacterium sp. VKM Ac-1815D mutant strain Et1 were shown to carry out 17beta-reduction, 17beta-dehydrogenation and 1(2)-reduction of 3-keto-C(19)-steroids. Two 17-hydroxy steroid dehydrogenases (17-OH SDH) were partially purified from the strain by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-sephacel and gel-filtration on Bio-Gel A. The enzymes differed in chromatographic properties and specific activities. One enzyme--17-OH SDH (2) (tetramer, M(r) approximately 210,000) was found to be responsible for bi-directional reduction-oxidation of steroids at C 17, whereas the other one--17-OH SDH (1) (monomer, M(r) approximately 68,000) specifically catalysed 17beta-dehydrogenation of 17-hydroxysteroids (testosterone and 1(2)-dehydro testosterone). The 17beta-reduction of 1-ene-17-ketosteroids was accompanied by 1(2)-reduction. A role of 1-ene-reductase as a steroid-binding protein associated with 17-OH SDH (2) in Mycobacterium sp. is discussed.

Localization of 17β-hydroxysteroid dehydrogenase in Mycobacterium sp. VKM Ac-1815D mutant strain

The localization of mycobacterial 17beta-hydroxysteroid dehydrogenase (17beta-OH SDH) was studied using cell fractionation and cytochemical investigation. Mycobacterium sp. Et1 mutant strain derived from Mycobacterium sp. VKM Ac-1815D and characterized by increased 17beta-OH SDH activity was used as a model organism. Subcellular distribution study showed both soluble and membrane-bound forms of mycobacterial 17beta-hydroxysteroid dehydrogenase. The cytochemical method based on a copper ferrocyanide procedure followed by electron microscopic visualization was applied in order to investigate the intracellular localization of bacterial 17beta-OH SDH in more detail. The enzyme was found to be located in the peripheral cytoplasmic zone adjoining the cytoplasmic membrane (CM). 17beta-OH SDH was loosely membrane bound and easily released into the environment under the cell integrity failure.

Conversion of 1-benzoylindole by Aspergillus strains

Abstract Biotransformation of 1-benzoylindole (BI) by the strains Aspergillus flavus VKM F-1024 and Aspergillus oryzae VKM F-44 was studied. The major metabolites isolated were identified as 4-hydroxyindole (4-HI), 5-hydroxyindole (5-HI), 4-hydroxy-1-benzoylindole, 4-hydroxy-1-(4′-hydroxy)-benzoylindole and indole. The structure of the metabolites was determined by mass spectrometry and proton nuclear magnetic resonance spectroscopy. The pathways of BI metabolism via initial monohydroxylation at C-4 and C-5 followed by cleavage of the benzoyl substituent to yield 4-HI and 5-HI were proposed. Indole was formed as a by-product, and its role as a potent inhibitor of BI hydroxylation at C-4 and C-5 is discussed.

Bioconversion of 6-(N-methyl-N-phenyl)aminomethyl androstane steroids by Nocardioides simplex

HIGHLIGHTSNocardioides simplex converts 6‐(N‐methyl‐N‐phenyl)aminomethyl androstanes.Only &bgr;‐stereoisomers undergo 1(2)‐dehydrogenation with N. simplex cells.(N‐methyl‐N‐phenyl)aminomethyl substitution at C6 prevents biodegradation of steroid core. ABSTRACT The newly synthesized (&agr;/&bgr;)‐diastereomers of 6‐(N‐methyl‐N‐phenyl)aminomethylandrost‐4‐ene‐3,17‐dione (5) and 6‐(N‐methyl‐N‐phenyl)aminomethylandrost‐4‐en‐17&bgr;‐ol‐3‐one (6) were firstly investigated as substrates for the whole cells of Nocardioides simplex VKM Ac‐2033D in comparison with their unsubstituted analogs, – androst‐4‐ene‐3,17‐dione (1) and androst‐4‐en‐17&bgr;‐ol‐3‐one (2). 1(2)‐Dehydroderivatives were identified as the major bioconversion products from all the substrates tested. When using the mixtures of (&agr;/&bgr;)‐stereoisomers of 5 and 6 as the substrates, only &bgr;‐stereoisomers of the corresponding 1,4‐diene‐steroids were formed. Along with 1(2)‐dehydrogenation, N. simplex VKM Ac‐2033D promoted oxidation of the hydroxyl group at C‐17 position of 6: both 6(&agr;) and 6(&bgr;) were transformed to the corresponding 17‐keto derivatives. No steroid core destruction was observed during the conversion of the 6‐substituted androstanes 5 and 6, while it was significant when 1 or 2 was used as the substrate. The results suggested high potentials of N. simplex VKM Ac‐2033D for the generation of novel 1(2)‐dehydroanalogs.