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PubMedDate not supplied

Cloning of cDNA encoding an NAD(+)-dependent isoform of 11 beta-hydroxysteroid dehydrogenase in sheep kidney.

11 beta-Hydroxysteroid dehydrogenase (11-HSD) catalyzes the conversion of cortisol to cortisone and corticosterone to 11-dehydrocorticosterone. This activity may be required to confer normal ligand specificity upon the mineralocorticoid receptor. Although an isozyme of 11-HSD was previously isolated from rat liver, a different isozyme is apparently expressed in mineralocorticoid target tissues. We isolated a sheep kidney cDNA clone encoding this isozyme by expression screening using Xenopus oocytes. The cDNA is 1.8 kb in length and encodes a protein of 427 amino acid residues with a predicted M(r) of 46,700. When expressed in oocytes, this enzyme functions as an NAD(+)-dependent 11 beta-hydrogenase with very high affinity for steroids, but it has no detectable reductase activity. It is 37% identical in amino acid sequence to an NAD(+)-dependent isozyme of 17 beta-hydroxysteroid dehydrogenase, but only 20% identical to the NADP(+)-dependent liver isozyme of 11-HSD. It is expressed at high levels in the kidney and adrenal and at lower levels in the colon. The corresponding gene is present in a single copy in the sheep genome. In humans, this gene is a candidate locus for the syndrome of apparent mineralocorticoid excess, a form of hypertension postulated to result from 11-HSD deficiency in mineralocorticoid target tissues.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMedDate not supplied

Cloning and expression of a novel tissue specific 17beta-hydroxysteroid dehydrogenase.

The 11beta-hydroxysteroid dehydrogenases (11betaHSD) modulate intracellular glucocorticoid levels, with 11betaHSD1 converting cortisone to cortisol mainly in the liver, and 11betaHSD2 performing the reverse reaction in sodium transporting epithelia and placenta. We have attempted to expand the 11betaHSD subfamily by isolating homologous cDNA's. Expressed Sequence Tag databases were screen with segments of the 11betaHSD1 enzyme amino acid sequence and Pan1b identified as a new member of the short chain alcohol dehydrogenase superfamily. Northern blot analysis of total RNA from human tissues showed a single band at 1.9 kb and a tissue specific pattern of expression with high levels in the liver, adrenal carcinoma, lung and small intestine, and much lower levels in the kidney, heart and placenta. Expression studies in a Chinese hamster ovary cell line (CHOP) showed that Pan1b did not metabolize glucocorticoids. However, preliminary studies on a range of substrates revealed that Pan1b acted as a dehydrogenase on 17beta-hydroxysteroids, although further kinetic analysis was confounded by large amounts of endogenous oxidoreductase activity in CHOP cells. These studies suggest the existence of a novel human 17betaHSD enzyme.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1983

Human placental 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase. Studies with 6 beta-bromoacetoxyprogesterone.

Two soluble enzyme activities, 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase, copurified from the cytosol fraction of human term placenta, were identically inactivated by 6 beta-bromoacetoxyprogesterone. This affinity alkylating steroid binds at the enzyme-active site (Km = 866 microM; Vmax = 0.073 mumol/min/mg). Enzyme inactivation by four concentrations of 6 beta-bromoacetoxyprogesterone (molar ratio of steroid to enzyme, 71/1 to 287/1) causes irreversible and time-dependent loss of both the 17 beta- and 20 alpha-activities according to first order kinetics and affirms that the alkylating steroid is an active site-directed inhibitor (KI = 2.7 X 10(-3) M; k3 = 1.6 X 10(-3) s-1). Affinity radioalkylation studies using 6 beta-[2'-14C]bromoacetoxyprogesterone indicate that 2 mol of steroid are bound to each mole of inactivated enzyme dimer (Mr = 68,000). Amino acid analyses of the acid hydrolysate of radioalkylated enzyme show that 6 beta-bromoacetoxyprogesterone carboxymethylates cysteine (56%), histidine (22%), and lysine (8%) residues in the active site. These results are identical with those reported for 2-bromo[2'-14C]acetamidoestrone methyl ether radioalkylation of purified "17 beta-estradiol dehydrogenase." The parallel inactivation of 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase by 6 beta-bromoacetoxyprogesterone further shows that both activities reside at a single enzyme-active site. The radioalkylation profile supports our proposed model of one enzyme-active site wherein the bound progestin and estrogen substrates are inverted, one relative to the other.

17-Hydroxysteroid Dehydrogenases↗
PubMed2000

CHILD syndrome caused by deficiency of 3beta-hydroxysteroid-delta8, delta7-isomerase.

CHILD (congenital hemidysplasia, ichthyosis, and limb defects) syndrome is a rare, usually sporadic disorder associated with unilateral distribution of ichthyosiform skin lesions, limb defects, punctate calcifications of cartilaginous structures, and visceral anomalies. CHILD syndrome shares some manifestations with X-linked dominant Conradi-Hünermann syndrome (CDPX2), although the skeletal defects and skin lesions in CDPX2 are bilateral and asymmetric. Because CDPX2 patients have abnormal 8-dehydrosterol metabolism caused by mutations in 3beta-hydroxysteroid-delta8,delta7-isomerase, we measured plasma sterols in a patient with CHILD syndrome and found levels of 8-dehydrocholesterol and 8(9)-cholestenol increased to the same degree as in CDPX2 patients. Subsequently, we identified a nonsense mutation in exon 3 of the patient's 3beta-hydroxysteroid-delta8,delta7-isomerase gene. We speculate that at least some cases of CHILD syndrome are allelic with CDPX2 caused by 3beta-hydroxysteroid-delta8,delta7-isomerase deficiency.

Abnormalities, Multiple↗
PubMed2001

Bacterial expression, purification, and characterization of rat hydroxysteroid sulfotransferase STa.

Hydroxysteroid (alcohol) sulfotransferase catalyzes numerous reactions that are important to our understanding of the metabolism of both endogenous steroids and exogenous alcohols. Here we report a method for prokaryotic expression and rapid purification of the recombinant hydroxysteroid sulfotransferase STa, a major isoform of hydroxysteroid sulfotransferase in the rat. The cDNA encoding STa was cloned into a pET-3c vector and expressed in Escherichia coli BL21 cells. After disruption of the cells by sonication, the enzyme was purified in one step by affinity chromatography on adenosine 3',5'-diphosphate-agarose. The purified recombinant STa had a relative molecular mass on SDS-PAGE that was identical with the native hepatic STa in rat liver. The expressed enzyme displayed similar substrate inhibition characteristics with dehydroepiandrosterone as have been noted previously with the native enzyme purified from rat liver. Furthermore, the catalytic efficiency in sulfation of 7-hydroxymethyl-12-methylbenz[a]anthracene, as well as the stereoselectivity of sulfation of the enantiomers of 1-phenyl-1-heptanol and 1-naphthyl-1-ethanol, catalyzed by the recombinant STa were consistent with characteristics of the STa isolated from rat liver.

Animals↗
PubMed1989

Cloning and sequence analysis of a rat liver cDNA encoding hydroxysteroid sulfotransferase.

Nothing has been known of the cDNAs encoding sulfotransferases (STs) that catalyze sulfation of steroids and xenobiotics. In the present study, a female Sprague-Dawley (SD) rat liver cDNA library was screened with rabbit anti-serum raised against hydroxysteroid ST a (STa) purified from female SD rat liver cytosol. The cDNA isolated from the library consisted of 1,028 base pairs which had an open reading frame of 852 base pairs encoding the entire rat ST subunit of 284 amino acids. The N-terminal amino acid sequences of STa and the rat liver hydroxysteroid ST, bile acid ST I, both elucidated previously by the chemical method, had a strong homology with that deduced from the cDNA. Northern blot analysis of total RNAs from female and male rat livers showed a marked sex difference (female much greater than male) in the expressed level of the mRNA for the predicted ST subunit protein. A remarkable sex difference (female much greater than male) was also observed by immuno-blot analysis in the level of the hydroxysteroid ST protein(s) cross-reacting with the anti-serum in the rat liver cytosols.

Amino Acid Sequence↗
PubMed1990

cDNA cloning of the hydroxysteroid sulfotransferase STa sharing a strong homology in amino acid sequence with the senescence marker protein SMP-2 in rat livers.

A cDNA encoding hydroxysteroid sulfotransferase a (STa), which catalyzes activation of carcinogenic polycyclic hydroxymethyl-arenes, was isolated from a lambda gtll cDNA expression library constructed from poly(A)+RNA of a female Sprague-Dawley (SD) rat liver. The cDNA, designated as ST-40, consisted of 1,015 base pairs which had an open reading frame of 852 base pairs encoding the entire rat STa subunit of 284 amino acids. The nucleotide base sequence of the ST-40 cDNA shared a strong homology of 94.4% with that of ST-20 cDNA encoding a hydroxysteroid ST which had been reported by us. The deduced amino acid sequence of STa had a homology of 73.7% with that of an SD rat liver senescence marker protein (SMP-2) consisting of 282 amino acid residues. However, STa was found to share a much stronger homology of 92% on the average with SMP-2 in their four specific regions corresponding to about 60% of the total sequences, indicating SMP-2 to be an isozyme of hydroxysteroid ST.

Amino Acid Sequence↗
PubMed1989

Sulphotransferase-mediated activation of the carcinogen 5-hydroxymethyl-chrysene. Species and sex differences in tissue distribution of the enzyme activity and a possible participation of hydroxysteroid sulphotransferases.

Sulphation of the carcinogen 5-hydroxymethyl-chrysene (5-HCR) to the active metabolite 5-HCR sulphate occurred at significant rates in all of hepatic cytosols prepared from the male and female experimental animals, rats, mice, guinea-pigs and hamsters. The 5-HCR-sulphating activity was also found in kidney cytosols of all the experimental animals used, while their activities were much less than those of hepatic cytosols. In the male mice, the enzyme activity of testis was higher than any other examined tissue. Small intestine and adrenal of male and female guinea-pigs had relatively high enzyme activities. Small enzyme activities were also found in a variety of extrahepatic tissues of some of these animals. Marked species and sex differences (female much greater than male in the rat and mouse) were observed in the hepatic enzyme activity. In the female rat liver which showed the highest 5-HCR-sulphating activity among the examined tissues of all the animals, a typical hydroxysteroid sulphotransferase inhibitor, dehydroepiandrosterone (DHA) sulphate (1 mM), potently and competitively inhibited the sulphation of 5-HCR as well as that of DHA, a typical substrate for hydroxysteroid sulphotransferases. On the contrary, the phenol sulphotransferase inhibitors, pentachlorophenol and 2,6-dichloro-4-nitrophenol, had only a little effect on these enzyme activities even at a concentration of 50 microM that showed a potent inhibition of the phenol sulphotransferase activity. These results suggest that 5-HCR be sulphated in the female rat liver by hydroxysteroid sulphotransferases, but not by phenol sulphotransferases.

Animals↗
PubMed1995

Effects of hypophysectomy and thyroxine on the expression of hepatic oestrogen, hydroxysteroid and phenol sulphotransferases.

Sulphation in rats, and other mammals, is carried out by a family of sulphotransferase isoenzymes, which can be further subdivided into oestrogen, hydroxysteroid and phenol sulphotransferases. We have examined the effects of hypophysectomy on the activity and expression of representative members of the three major sulphotransferase sub-families in male Wistar rat liver cytosols, and have found that the different sub-families are subject to differential regulation by pituitary hormones. Our data show that in male rat liver hydroxysteroid sulphotransferases activity was increased, oestrogen sulphotransferases activity was not altered and phenol sulphotransferases activity was reduced. Further, we have studied the effect on sulphotransferase expression of administration of thyroxine and dexamethasone to hypophysectomized rats. Treatment of hypophysectomized rats with thyroxine virtually abolished oestrogen sulphotransferase activity in male rat liver but had no effect on hydroxysteroid sulphotransferase or phenol sulphotransferase activity. Treatment of hypophysectomized rats with dexamethasone had no effect on sulphotransferase activities. Quantitative immunoblot analysis of liver cytosols showed that these changes in enzyme activity were related to changes in levels of the respective enzyme proteins.

Animals↗
PubMed1990

Characterization of the steroid-metabolizing capacity of the hepatic cytochrome P450IIC5 expressed in COS-1 cells: 3 beta-hydroxysteroid dehydrogenase/delta 5----4 isomerase type activity.

Cytochrome P450IIC5 (rabbit liver 21-hydroxylase) is unusual among hepatic forms of cytochromes P450 because it catalyzes the conversion of one active steroid hormone (progesterone) to another active hormone (deoxycorticosterone). Another interesting aspect of this steroid-hydroxylating enzyme is the ability to convert delta 5-3 beta-hydroxysteroids to the delta 4-3-ketosteroid configuration. The delta 5-3-beta-hydroxysteroid, pregnenolone, was readily 21-hydroxylated, and this product was further metabolized to the delta 4-3-ketosteroid, deoxycorticosterone. It is suggested that the mechanism of this cytochrome P450-mediated, 3 beta-hydroxysteroid dehydrogenase/delta 5----4 isomerase-like reaction is through a gem-diol formation. In this study, COS-1 cells were transfected with the plasmid encoding cytochrome P450IIC5 to express a functional enzyme within the cell milieu. Transfected COS cells preferentially metabolize pregnenolone compared with all other steroids tested. Progesterone and 17 alpha-hydroxypregnenolone are also 21-hydroxylated, whereas 17 alpha-hydroxyprogesterone is a poor substrate. Substrate preference of this 21-hydroxylase differs from that seen with bovine adrenal P450XXIA1 (formerly P450C21) hydroxylase. Additionally, this study demonstrated that C19 steroids, like dehydroepiandrosterone and androstenedione, are hydroxylated at the 16 alpha position. Contrary to previous reports, no metabolite of estradiol-17 beta was detected, presumably due to the unstable nature of catechol estrogens (2-hydroxyestradiol).

Animals↗
PubMed2002

Teleost ovarian carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase: potential role in the production of maturation-inducing hormone during final oocyte maturation.

17alpha,20beta-Dihydroxy-4-pregnen-3-one is the major oocyte maturation-inducing hormone of several teleost species. Gonadotropin-induced increase in ovarian 20beta-hydroxysteroid dehydrogenase activity is essential for the synthesis of maturation-inducing hormone. Cloning and expression studies suggest that ayu (Plecoglossus altivelis) ovarian carbonyl reductase can function as 20beta-hydroxysteroid dehydrogenase. The amino acid sequence deduced from the isolated cDNA had 276 amino acid residues and shared approximately 60% homology with mammalian and teleostean carbonyl reductases. The sequence data search showed that the ayu cDNA clone belongs to the short-chain dehydrogenase/reductase family. The clear lysate prepared from Escherichia coli harboring the cDNA catalyzed the production of maturation-inducing hormone. Its identification was confirmed by two-dimensional, thin-layer chromatography followed by recrystallization. Purification of the E. coli-expressed cDNA product revealed that it possessed both carbonyl reductase and steroid dehydrogenase activities, and 17alpha-hydroxyprogesterone, the endogenous immediate precursor of maturation-inducing hormone, was one of the preferred substrates. Furthermore, Northern blot analysis denoted that the transcripts are present both in fully grown, immature ovarian follicles and at higher levels in mature ovarian follicles. These results demonstrate that the carbonyl reductase of ayu ovary is involved in the production of maturation-inducing hormone, and they provide evidence for a novel physiological role of this enzyme in the final maturation of oocytes. Based on its functional properties, the enzyme can be referred to as carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase.

Alcohol Oxidoreductases↗
PubMed2003

Conservation of the hydroxysteroid sulfotransferase SULT2B1 gene structure in the mouse: pre- and postnatal expression, kinetic analysis of isoforms, and comparison with prototypical SULT2A1.

A novel mouse hydroxysteroid sulfotransferase cDNA has been cloned, and organization of its gene structure has been determined. The new mouse sulfotransferase, SULT2B1a, and its closely related isoform, SULT2B1b, are derived from a single SULT2B1 gene as a result of an alternative exon I and differential splicing. Thus, the only structural distinction between the two SULT2B1 isoforms is at their amino-terminal ends. Importantly, in contrast to the prototypical mouse hydroxysteroid sulfotransferase SULT2A1, the SULT2B1 isoforms have a predilection for cholesterol. Real-time RT-PCR reveals that the SULT2B1a isoform is most abundantly expressed in the brain and spinal cord, whereas SULT2B1b and SULT2A1 are weakly, if at all, expressed in the central nervous system. On the other hand, the SULT2B1b isoform is the most prominent hydroxysteroid sulfotransferase expressed in skin, whereas SULT2A1 is strikingly expressed in the liver. The substrate specificities and differential expression patterns of the three SULT2 isozymes strongly suggest that they have distinct biologic roles to play. Of further interest, the mouse SULT2B1 and SULT2A1 genes are differentially expressed during embryonic development, with the former being expressed at all stages from E8.5-E19, whereas the latter is not expressed until E19. It is speculated that, during embryonic development, SULT2B1b is required for production of cholesterol sulfate essential for normal skin development, whereas SULT2B1a produces pregnenolone sulfate, an essential neurosteroid during development of the central nervous system.

Amino Acid Sequence↗
PubMed1993

Enzyme characteristics of two distinct forms of mouse 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase complementary deoxyribonucleic acids expressed in COS-1 cells.

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-Isomerase (3 beta HSD) catalyzes the conversion of delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids, an essential step in the biosynthesis of all biologically active steroid hormones. We previously reported the isolation of three distinct mouse cDNAs for 3 beta HSD (3 beta HSD I, II, and III) and tissue-specific expression of their mRNAs. 3 beta HSD I is expressed only in gonads and adrenal glands, and 3 beta HSD II and III are expressed in both liver and kidneys. In the current study, we present data which demonstrate that transiently expressed 3 beta HSD I and 3 beta HSD III proteins can catalyze the conversion of the delta 5-steroids, pregnenolone and dehydroepiandrosterone, to their respective delta 4-3-ketosteroids, progesterone and androstenedione. They also can dehydrogenate the 3 beta-hydroxy group of the 5 alpha-reduced steroid 5 alpha-androstanediol to yield dihydrotestosterone in the presence of the cofactor NAD+. The Km values of the expressed 3 beta HSD I (for each of these substrates) were all below 0.2 microM. Km values of 3 beta HSD III were greater for all substrates, with the greatest increase observed for pregnenolone, which was over 10-fold greater. Both forms of expressed protein can catalyze the reduction of dihydrotestosterone to 5 alpha-androstanediol in the presence of the cofactor NADH, but with considerably higher Km values (5.5 microM for form I and 6.8 microM for form III). The observed maximum velocity of form I was much higher for all substrates examined. RNase protection and immunoblot analysis of expressed 3 beta HSD I and III indicate that the difference in maximum velocity reflect differences in the steady state levels of mRNA and amounts of protein. In addition, the expressed 3 beta HSD III protein analyzed by Western blot has a lower mobility than the 3 beta HSD I protein, both similar in mol wt to the 3 beta HSD proteins detected in mouse liver and adrenal glands, respectively. These data demonstrate that an isoform of 3 beta HSD expressed in liver and kidney has the capacity to convert delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids. The data suggest that a homologous human 3 beta HSD isoform could play an important role in cases of genetic deficiency of the gonadal and adrenal isoform.

Androstane-3,17-diol↗
PubMed1993

Estrogen and hydroxysteroid sulfotransferases in guinea pig adrenal cortex: cellular and subcellular distributions.

This report describes for the first time the cellular and subcellular localization of estrogen sulfotransferase (EST) as well as the subcellular localization of hydroxysteroid sulfotransferase (HST) in the mammalian adrenal cortex. A 34-kilodalton EST and two HSTs with 3 alpha- and 3 beta-hydroxysteroid substrate specificities (32 and 33 kilodaltons, respectively) were previously purified from guinea pig adrenal cortex and characterized. Western blots were used to establish that two antisera generated against EST were highly specific for EST, whereas three antisera generated against the HSTs were highly specific for the HSTs, but did not distinguish between the 3 alpha- and 3 beta HSTs. Light and electron microscopic immunoperoxidase labeling with these antisera revealed that the sulfotransferases were expressed only within the ACTH-responsive layers of the guinea pig adrenal cortex, with EST localized to zona fasciculata and zona reticularis cells, and the HSTs confined to the zona reticularis. No labeling was detected in the zona glomerulosa or adrenal medulla. EST was concentrated in cell nuclei; sparse labeling was distributed throughout the cytoplasm. HST labeling was intense in smooth endoplasmic reticulum of zona reticularis cells, but was absent from nuclei. Ovoid inclusions about 1-4 microns in diameter, with no limiting membrane, were observed in zona reticularis cells; these inclusions were strongly labeled for both EST and HSTs. No gender-specific differences in distribution or labeling intensity were apparent. The high concentration of EST immunoreactivity in nuclei suggests that EST may play a role in modulating the ability of active estrogens to regulate gene expression in ACTH-responsive cells. The distribution of HST labeling suggests that sulfonation of adrenocortical 3-hydroxysteroids takes place largely within smooth endoplasmic reticulum in the zona reticularis in adult guinea pigs.

Adrenal Cortex↗
PubMed1991

20 beta-hydroxysteroid dehydrogenase of neonatal pig testis: reverse catalytic (oxidation) reaction.

Neonatal pig testicular 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) catalyzed the oxidation of 20 beta-hydroxysteroids, 17 alpha,20 beta-dihydroxypregn-4-en-3-one and 20 beta-hydroxypregn-4-en-3-one in the presence of beta-nicotinamide adenine dinucleotide phosphate (beta-NADP+). The behavior of 20 beta-HSD activity toward the substrate of 17 alpha,20 beta-dihydroxypregn-4-en-3-one differed from the catalytic reaction for 20 beta-hydroxypregn-4-en-3-one. The enzyme could catalyze not only 20 beta-hydroxysteroids but also 20 alpha-hydroxy-5-ene steroids, 20 alpha-hydroxypregn-5-en-3 beta-ol and 17 alpha,20 alpha-hydroxypregn-5-en-3 beta-ol with 22.1 and 8.7% of activity relative to 20 beta-hydroxypregn-4-en-3-one, respectively. The enzyme preferentially required beta-NADP+, and also utilized beta-nicotinamide adenine dinucleotide beta-NAD+ and beta-nicotinamide adenine dinucleotide 3'-phosphate (beta-3'-NADP+) nonspecifically as the cofactor. The optimum pH was observed at pH 7.5 with the substrate of 20 beta-hydroxypregn-4-en-3-one. The activation energies obtained from oxidation-reduction reactions of 20 beta-HSD for the substrate of 20 beta-hydroxypregn-4-en-3-one, progesterone and 17 alpha-hydroxyprogesterone were estimated at 13.8, 27.0 and 20.0 kcal/mol, respectively.

Animals↗
PubMed1998

20beta-hydroxysteroid dehydrogenase catalyzes ketone-reduction of acetohexamide, an oral antidiabetic drug, in liver microsomes of adult male rats.

We examined the catalytic properties and physiological function of an enzyme responsible for the ketone-reduction of acetohexamide, an oral antidiabetic drug, in liver microsomes of adult male rats. Progesterone, 17alpha-hydroxyprogesterone, cortisone and cortisol, which have a ketone group at 20-position of C21-steroids, were potent inhibitors for ketone-reduction of acetohexamide in liver microsomes of adult male rats. Progesterone was also found to inhibit competitively the ketone-reduction of acetohexamide, suggesting that the ketone-reduction of acetohexamide and progesterone is catalyzed by the same enzyme. When progesterone was used as a substrate, 20beta-hydroxysteroid dehydrogenase present in liver microsomes of adult rats, such as acetohexamide reductase, exhibited a male-specific and androgen-dependent activity. Furthermore, a significant correlation was observed between the activities of 20beta-hydroxysteroid dehydrogenase and acetohexamide reductase in liver microsomes of individual male rats at various ages. Based on all results, we conclude that 20beta-hydroxysteroid dehydrogenase catalyzes the ketone-reduction of acetohexamide in liver microsomes of adult male rats.

Acetohexamide↗
PubMed2000

Functional expression, purification, and characterization of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.

3alpha-Hydroxysteroid dehydrogenase (3alpha-HSD) catalyzes the oxidoreduction at carbon 3 of steroid hormones and is postulated to initiate the complete mineralization of the steroid nucleus to CO(2) and H(2)O in Comamonas testosteroni. By this activity, 3alpha-HSD provides the basis for C. testosteroni to grow on steroids as sole carbon and energy source. 3alpha-HSD was cloned and overexpressed in E. coli and purified to homogeneity by an affinity chromatography system as His-tagged protein. The recombinant enzyme was found to be functional as oxidoreductase toward a variety of steroid substrates, including androstanedione, 5alpha-dihydrotestosterone, androsterone, cholic acid, and the steroid antibiotic fusidic acid. The enzyme also catalyzes the carbonyl reduction of nonsteroidal aldehydes and ketones such as metyrapone, p-nitrobenzaldehyde and a novel insecticide (NKI 42255), and, based on this pluripotent substrate specificity, was named 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR). It is suggested that 3alpha-HSD/CR contributes to important defense strategies of C. testosteroni against natural and synthetic toxicants. Antibodies were generated in rabbits against the entire 3alpha-HSD/CR protein, and may now be used for evaluating the pattern of steroid induction in C. testosteroni on the protein level. Upon gel permeation chromatography the purified enzyme elutes as a 49.4 kDa protein revealing for the first time the dimeric nature of 3alpha-HSD/CR of C. testosteroni.

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

Expression in Escherichia coli and characterization of a bile acid-inducible 3 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708.

We have previously cloned and sequenced three members of a bile acid-inducible gene family from Eubacterium sp. strain VPI 12708 that encode 27,000-M(r) polypeptides. Two copies of these genes (baiA1 and baiA3) are identical, while the third copy (baiA2) encodes a polypeptide sharing 92% amino acid identity with the baiA1 and baiA3 gene products. We have overexpressed the baiA1 gene in Escherichia coli and analyzed the expressed activity. Thin-layer chromatography of 14C-labeled bile acid products from reactions using cell-free extracts revealed a 3 alpha-hydroxysteroid dehydrogenase activity for the BaiA1 protein. The BaiA1 protein could utilize both NAD+ and NADP+, and the preferred steroid substrate was the cholyl-coenzyme A conjugate rather than free cholic acid. These results show that the BaiA proteins are novel 3 alpha-hydroxysteroid dehydrogenases.

3-Hydroxysteroid Dehydrogenases↗