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At least 19 recordsLinked to original sources
PubMed1995

Molecular cloning, expression in Streptomyces lividans, and analysis of a gene cluster from Arthrobacter simplex encoding 3-ketosteroid-delta 1-dehydrogenase, 3-ketosteroid-delta 5-isomerase and a hypothetical regulatory protein.

The Arthrobacter simplex gene coding for 3-ketosteroid-delta 1-dehydrogenase, a key enzyme in the degradation of the steroid nucleus, was cloned in Streptomyces lividans. Nucleotide sequence analysis revealed that the gene for 3-ketosteroid-delta 1-dehydrogenase (ksdD) is clustered with at least two more genes possibly involved in steroid metabolism. Upstream of ksdD, we found a gene, ksdR, encoding a hypothetical regulatory protein that shows homologies to KdgR, the negative regulator of pectin biodegradation in Erwinia, and GyIR, the activator for glycerol metabolism in Steptomyces. A helix-turn-helix DNA-binding domain can be predicted at similar positions near the N-terminal of KsdR, KdgR and GyIR. ksdl adjoining downstream to ksdD codes for a protein that has strong similarities to 3-ketosteroid-delta 5-isomerases. The highly conserved Tyr and Asp residues are present in the active-centre motif of the enzyme. The translated ksdD gene product was found to be similar to the 3-ketosteroid-delta 1-dehydrogenase of Pseudomonas testosteroni and to the fumarate reductase of Shewanella putrefaciens. A region highly conserved between the two steroid dehydrogenases can be aligned to the active-centre motif of the fumarate reductase. S. lividans strains carrying the ksdD gene overexpressed 3-ketosteroid-delta 1-dehydrogenase. The expression of 3-ketosteroid-delta 5-isomerase, however, was barely detectable in recombinant S. lividans strains carrying the ksdl gene, or in the parental Arthrobacter strain.

Amino Acid Sequence↗
PubMed1982

Inactivation of delta5-3-ketosteroid isomerase(s) from beef adrenal cortex by beta, gamma-acetylenic ketosteroids.

The beta, gamma-acetylenic ketosteroids, 5-10-seco-19-norpregn-5-yne-3,-10,20-trione approximately 1 and 5,10-secoestr-5-yne-3,10,17-trione approximately 2 irreversibly inactivate both the C19-and the C21-delta 5-3-ketosteroid isomerase activities of beef adrenal cortex microsomes. At saturating concentrations of inhibitor half-lives of these enzyme activities vary from 45 to 240 s. It is uncertain whether the enzyme generates its own alkylating agent by isomerizing compounds approximately 3 and approximately 4 to the corresponding allenic ketones, namely (4R)-5,10-seco-19-norpregn-4,5-diene-3,10,20-trione approximately 3 and (4R)-5,10-secoestra-4,5-diene-3,10,17-trione approximately 4 since these are formed spontaneously in the buffer used to stabilize enzyme activity. In the presence of catalytic quantities of adrenal enzyme compound approximately 4 is a powerful competitive inhibitor for both 5-androstene-3,17-dione (Ki 8.0 microM) and 5-pregnene-3,20-dione (Ki 3.5 microM) indicating that the eventual alkylating event is active site-directed. The differences in Ki values and half-lives for inactivation support the view that the C19- and C21-delta 5-3-ketosteroid isomerase activities do not reside at the same catalytic site in beef adrenal cortex microsomes.

Adrenal Cortex↗
PubMed1995

Enzymatic and nonenzymatic polarizations of alpha,beta-unsaturated ketosteroids and phenolic steroids. Implications for the roles of hydrogen bonding in the catalytic mechanism of delta 5-3-ketosteroid isomerase.

Ketosteroids (e.g., 19-nortestosterone) and phenolic steroids (e.g., 17 beta-estradiol and 17 beta-dihydroequilenin), which are potent competitive inhibitors of delta 5-3-ketosteroid isomerase (isomerase, EC 5.3.3.1) of Pseudomonas testosteroni, undergo significant polarization upon binding to the active site of the enzyme. The 10 nm red shift of the UV absorption maximum of the enone chromophore of 19-nortestosterone, which occurs in the enzyme-steroid complex, resembles that observed when this steroid is exposed to strong acid. The UV and fluorescence spectral changes of 17 beta-estradiol and 17 beta-dihydroequilenin in the enzyme-bound complex resemble the spectra of ionized phenolate species in aqueous basic solutions. Since most enzymes bind their substrates and competitive inhibitors in a solvent-inaccessible hydrophobic environment, and the generation of charges in such nonpolar environments is unfavorable, we investigated the possibility that the spectral perturbations of the steroids might arise from strong hydrogen bonding in nonpolar environments. For this purpose, the spectral properties of model compounds capable of forming intramolecular hydrogen bonds were studied in nonpolar solvents. Thus, 4-hydroxyandrost-4-ene-3,17-dione, in which the 4-hydroxyl group is intramolecularly hydrogen-bonded to the 3-carbonyl group through a five-membered ring, exhibits a lambda max of 276.0 nm, while the corresponding 4-methyl ether, 4-methoxyandrost-4-ene-3,17-dione, which cannot form an internal hydrogen bond, shows a lambda max of 258.5 nm in aqueous solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Androstenedione↗
PubMed1975

Chemical modification of amino acid residues associated with the delta-4-3-ketosteroid-dependent photoinactivation of delta-5-3-ketosteroid isomerase.

The photoinactivation of the Δ (5)-3-ketosteroid isomerase of Pseudomonas testosteroni in the presence of 3-oxo-4-estren-17β-yl acetate and air is accompanied by destruction of histidine and aspartate (or asparagine). The first order rate constant of photoinactivation of the enzyme is equal, within experimental error, to the first order rate constant for the destruction of a single aspartate (or asparagine) residue and is considerably greater than the first order rate constant for the destruction of a single residue of histidine. When the photolysis is carried out under anaerobic conditions, only aspartic acid (or asparagine) is destroyed as enzyme is inactivated. Both inactivation and aspartate (or asparagine) destruction occur to a greater extent in the absence of oxygen than in its presence. The destruction of histidine, on the other hand, is found to be strictly oxygen-dependent. These results suggest that photochemical modification of a single residue of aspartate (or asparagine) is largely, if not entirely, responsible for photoinactivation of the enzyme under these conditions. When irradiated in the presence of 3-oxo-4-entren-17β-yl acetate, performic acid-oxidized bovine pancreatic ribonuclease does not suffer any detectable destruction of its aspartic or asparaginyl residues but does undergo significant destruction of its histidine residues. These observations suggest that the aspartate (or asparagine) residue modification found with isomerase is an active site-directed photochemical reaction, whereas the modification of histidine may not be.

Amino Acids↗
PubMed1994

Measurement of urinary androgen sulfates without previous hydrolysis: a tool to investigate adrenarche. Determination of total 17-ketosteroid sulfates.

According to published data the group of urinary total 17-ketosteroid sulfates appears to represent an index of overall adrenal androgen production, at least before the onset of puberty. To quantify total 17-ketosteroid sulfates a modified colorimetric assay based on the Zimmermann reaction was validated. 17-ketosteroid sulfates were measured without previous hydrolysis (as conjugated Zimmermann chromogens against authentic dehydroepiandrosterone sulfate (DHEAS) as assay standard) after C18 reversed-phase extraction and LH-20 chromatography. Intra- and inter-assay coefficients of variation were 8.4% (15.0%) and 5.9% (17.6%), respectively, at urinary 17-ketosteroid sulfate concentrations of 10.8 (1.9) nmol/ml. Recoveries observed in spiking and parallelism experiments varied between 88 and 102%. In a group of 4-year-old children showing a renal DHEAS output of less than 0.1 mumol/d/1.73 m2 (measured by radioimmunoassay) a relatively high median 17-ketosteroid sulfate excretion of 1.29 mumol/d/1.73 m2 was found. Older children aged 8 years as well as a group aged 12-14 years demonstrated only moderately higher urinary 17-ketosteroid sulfates whereas excretion of DHEAS/d/1.73 m2 more than tripled from age group to age group. For children from 8 years onwards, adolescents, and adults, linear regression analysis indicated that urinary DHEAS elevations seem to contribute with a constant proportion of approximately 70% to the increments of total urinary 17-ketosteroid sulfates. These findings suggest that the attainment of such a constant relationship (between the total 17-ketosteroid sulfates and their major component) from about 8 years of age onwards could represent the hormonal correlate of the completion of the continuous zona reticularis in the adrenal gland (developing around this age from a focal reticularis zone).

17-Ketosteroids↗
PubMed1990

Purification and characterization of 3-ketosteroid-delta 1-dehydrogenase from Nocardia corallina.

The inducible 3-ketosteroid-delta 1-dehydrogenase of Nocardia corallina which catalyzes the introduction of a double bond into the position of carbon 1 and 2 of ring A of 3-ketosteroid has been obtained in four steps with a 50% yield and 360-fold purification. The enzyme is homogeneous as judged by SDS-gel electrophoresis and is a monomeric protein with a molecular weight of 60,500. The isoelectric point of the enzyme is about 3.1. The enzyme contains 1 mol of flavin adenine dinucleotide per mol of protein, and has a typical flavoprotein absorption spectrum with maxima of 458, 362 and 268 nm. The enzyme is very stable in the absence of added cofactors, and catalyzes the dehydrogenation of delta 4-3-ketosteroids in the presence of phenazine methosulfate, which acts as an excellent electron acceptor. Potassium ferricyanide and cytochrome c did not act as electron acceptors. The delta 1-dehydrogenation was also stimulated by molecular oxygen with stoichiometric production of hydrogen peroxide and delta 1,4-3-ketosteroid. The optimum pH is 10 for dehydrogenation using phenazine methosulfate, and is between 8.5 and 10 for the oxidase reaction. The enzyme oxidizes a wide variety of 3-ketosteroids, but not 3 beta-hydroxysteroids. 3-Ketosteroids having an 11 alpha- or 11 beta-hydroxyl group were oxidized at slow rates. The purified enzyme catalyzes efficiently aromatization of the A-ring of 19-nortestosterone and 19-norandrostenedione to produce estradiol and estrone. 19-Hydroxytestosterone, 19-hydroxyandrostenedion and 19-oxotestosterone were converted to the respective phenolic steroids with cleavage of the C10 side-chain. Activities of 3-ketosteroid-delta 4-dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, 3 beta-hydroxysteroid dehydrogenase and 17 beta-hydroxysteroid dehydrogenase were not observed in the purified preparations. Properties of this novel flavoprotein enzyme are discussed.

Amino Acids↗
PubMed1992

Human ovarian 17-ketosteroid oxidoreductase: unique characteristics of the granulosa-luteal cell and stromal enzyme.

OBJECTIVES: We attempted to test the hypothesis that distinct forms of the 17-ketosteroid oxidoreductase exist in the human ovary and to compare its activity in stroma obtained from normally cycling women and from hyperandrogenic women. STUDY DESIGN: Human ovarian granulosa-luteal cell and stromal 17-ketosteroid oxidoreductase were examined in cell incubations and subcellular homogenates. RESULTS: In subcellular homogenates of granulosa-luteal cells 17-ketosteroid oxidoreductase activity was greater in the cytosol fraction than in the membrane fraction. In contrast, in homogenates of both ovarian stroma and Leydig cells its activity was greater in the membrane fraction than in the cytosol fraction. At the substrate concentrations used estrone was a better substrate than androstenedione for the granulosa-luteal cell 17-ketosteroid oxidoreductase. In contrast, androstenedione was a better substrate than estrone for that in ovarian stromal and Leydig cell membranes. In incubations of ovarian stroma from hyperandrogenic women, significantly more testosterone accumulated in the medium per milligram of tissue than in the medium of incubations of ovarian stroma from normally cycling women (142 +/- 48 vs 7.9 +/- 7.5 pg testosterone per milligram of tissue per 48 hours, mean +/- SD, p less than 0.05). The ratio of testosterone to androstenedione was significantly higher in the medium of incubations of ovarian stroma from hyperandrogenic women than in that from normally cycling women (0.61 vs 0.25, mean, p less than 0.05). The ratio of serum testosterone to androstenedione was significantly greater in hyperandrogenic women than in normally cycling control women (0.31 +/- 0.11 vs 0.20 +/- 0.03, mean +/- SD, p less than 0.05). CONCLUSION: The localization (cytosol fraction) and substrate specificity (estrone) of the granulosa-luteal cell 17-ketosteroid oxidoreductase enzyme resembles that seen in human placenta. The localization (membrane fraction) and substrate specificity (androstenedione) of the ovarian stromal 17-ketosteroid oxidoreductase enzyme resembles that seen in Leydig cells. It may be one enzyme that exists in multiple forms or it may be two (or more) enzymes. In some hyperandrogenic women the ovarian stromal 17-ketosteroid oxidoreductase may be more active than in normally cycling women, contributing to an abnormally increased testosterone production rate.

17-Hydroxysteroid Dehydrogenases↗