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(1/27) Isoleucine-15 of rainbow trout carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase is critical for coenzyme (NADPH) binding.

Carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase (CR/20beta-HSD) is an enzyme that converts 17alpha-hydroxyprogesterone to 17alpha, 20beta-dihydroxy-4-pregnen-3-one (the maturation-inducing hormone of salmonid fish). We have previously isolated two types of CR/20beta-HSD cDNAs from ovarian follicle of rainbow trout (Oncorhynchus mykiss). Recombinant proteins produced by expression in Escherichia coli in vitro showed that one (type A) had CR and 20beta-HSD activity but that the other (type B) did not. Among the three distinct residues between the protein products encoded by the two cDNAs, two residues (positions 15 and 27) are located in the N-terminal Rossmann fold, the coenzyme binding site. To investigate the structure/function relationships of CR/20beta-HSDs, we generated mutants by site-directed mutagenesis at the following positions: MutA/I15T, MutB/T15I, and MutB/Q27K. Enzyme activity of wild-type A was abolished by substitution of Ile-15 by Thr (MutA/I15T). Conversely, enzyme activity was acquired by the replacement of Thr-15 with Ile in type B (MutB/T15I). MutB/T15I mutant showed properties similar to the wild-type A in every aspect tested. Mutation MutB/Q27K had only partial enzyme activity, indicating that Ile-15 plays an important role in enzyme binding of cofactor NADPH.  (+info)

(2/27) Affinity labeling of steroid binding sites. Study of the active site of 20beta-hydroxysteroid dehydrogenase with 2alpha-bromoacetoxyprogesterone and 11alpha-bromacetoxyprogesterone.

To further characterize the active site of 20beta-hydroxysteroid dehydrogenase (EC 1.1.1.53) from Streptomyced hydrogenans we synthesized 2alpha-bromoacetoxyprogesterone, a substrate for the enzyme in 0.05 M phosphate buffer at 25 degrees, pH 7.0, with Km and Vmax values of 1.90 X 10(-5) M and 6.09 nmol/min/mg of enzyme, respectively. This affinity labeling steroid inactivates 20beta-hydroxysteroid dehydrogenase in an irreversible and time-dependent manner which follows pseudo-first order kinetics with a t1/2 value of 4.6 hours. 2alpha-[2-3H]Bromoacetoxyprogesterone was synthesized and used to radiolabel the enzyme active site. Amino acid analysis of the acid hydrolysate of the radiolabeled enzyme supports a mechanism whereby the steroid moiety delivers the alkylating group to the steroid binding site of the enzyme where it reacts with a methionyl residue. Both 2alpha- and 11alpha-bromoacetoxyprogesterone alkylate a methionyl residue at the active site of 20beta-hydroxysteroid dehydrogenase. The enzyme was inactivated with a mixture containing both 2alpha-[2-3H]Bromoacetoxyprogesterone and 11alpha-2[2-14C]bromoacetoxyprogesterone. Following degradation of separate aliquots of the radiolabeled enzyme by cyanogen bromide or trypsin, the protein fragments were separated by gel filtration and ion exchange chromatography. Resolution of peptides carrying the 3H label from those possessing the 14C label demonstrates that 2alpha-bromoacetoxyprogesterone and 11alpha-bromoacetoxyprogesterone each label a different methionine at the steroid binding site of 20beta-hydroxysteroid dehydrogenase.  (+info)

(3/27) 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.  (+info)

(4/27) Ovarian carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase shows distinct surge in messenger RNA expression during natural and gonadotropin-induced meiotic maturation in nile tilapia.

Meiotic maturation in fish is accomplished by maturation-inducing hormones. 17alpha,20beta-Dihydroxy-4-pregnen-3-one (17alpha,20beta-DP) was identified as the maturation-inducing hormone of several teleosts, including Nile tilapia. A cDNA encoding 20beta-hydroxysteroid dehydrogenase (20beta-HSD), the enzyme that converts 17alpha-hydroxyprogesterone to 17alpha,20beta-DP, was cloned from the ovarian follicle of Nile tilapia. Genomic Southern analysis indicated that 20beta-HSD probably exists as a single copy in the genome. The Escherichia coli-expressed cDNA product oxidized both carbonyl and steroid compounds, including progestogens, in the presence of NADPH. Carbonyl reductase-like 20beta-HSD is broadly expressed in various tissues of tilapia, including ovary, testis, and gill. Northern blot and reverse transcription polymerase chain reaction analyses during the 14-day spawning cycle revealed that the expression of 20beta-HSD in ovarian follicles is low from Day 0 to Day 8 after spawning and is not detectable on Day 11. Distinct expression was evident at Day 14, the day of spawning. In males, 20beta-HSD expression was observed continually in mature testes but not in immature testes of 30-day-old fish. In vitro incubation of postvitellogenic immature follicles (corresponding to Day 11 after spawning) with hCG induced the expression of 20beta-HSD mRNA transcripts within 1-2 h, followed by the final meiotic maturation of oocytes. In tissues such as gill, muscle, brain, and pituitary, however, hCG treatment did not induce any changes in the levels of mRNA transcripts. Actinomycin D blockade of hCG-induced 20beta-HSD expression and final oocyte maturation demonstrated the involvement of transcriptional factors. The carbonyl reductase-like 20beta-HSD plays an important role in the meiotic maturation of tilapia gametes.  (+info)

(5/27) An aldose reductase with 20 alpha-hydroxysteroid dehydrogenase activity is most likely the enzyme responsible for the production of prostaglandin f2 alpha in the bovine endometrium.

Prostaglandins are important regulators of reproductive function. In particular, prostaglandin F2 alpha (PGF(2 alpha)) is involved in labor and is the functional mediator of luteolysis to initiate a new estrous cycle in many species. These actions have been extensively studied in ruminants, but the enzymes involved are not clearly identified. Our objective was to identify which prostaglandin F synthase is involved and to study its regulation in the endometrium and in endometrial primary cell cultures. The expression of all previously known prostaglandin F synthases (PGFSs), two newly discovered PGFS-like genes, and a 20 alpha-hydroxysteroid dehydrogenase was studied by Northern blot and reverse transcription PCR. These analyses revealed that none of the known PGFS or the PGFS-like genes were significantly expressed in the endometrium. On the other hand, the 20 alpha-hydroxysteroid dehydrogenase gene was strongly expressed in the endometrium at the time of luteolysis. The corresponding recombinant enzyme has a K(m) of 7 microM for PGH(2) and a PGFS activity higher than the lung PGFS. This enzyme has two different activities with the ability to terminate the estrous cycle; it metabolizes progesterone and synthesizes PGF(2 alpha). Taken together, these data point to this newly identified enzyme as the functional endometrial PGFS.  (+info)

(6/27) Pig testicular 20 beta-hydroxysteroid dehydrogenase exhibits carbonyl reductase-like structure and activity. cDNA cloning of pig testicular 20 beta-hydroxysteroid dehydrogenase.

cDNA inserts encoding 20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53) were, for the first time, isolated and cloned from a pig testis cDNA library using synthetic oligonucleotides deduced from the partially determined amino acid sequences. The cDNA contains an open reading frame predicted to encode 289 amino acid residues. Surprisingly, it has 85% amino acid homology to human carbonyl reductase. The purified enzyme exhibited carbonyl reductase activity. Adenine-rich sequence was located in the 3'-untranslated nine-rich sequence was located in the 3'-untranslated region, which may mean that the gene originates by retroposition. RNA transcripts of 1.3, 3, and 6 kilobases were detected in poly(A)+ RNA extracted from pig testis by Northern blot hybridization. The steady-state level of the RNA species increased to a maximum in testes from 10-day-old pigs, but rapidly declined thereafter to the same levels found in testes of mature animals.  (+info)

(7/27) Short-chain dehydrogenases. Proteolysis and chemical modification of prokaryotic 3 alpha/20 beta-hydroxysteroid, insect alcohol and human 15-hydroxyprostaglandin dehydrogenases.

Prokaryotic 3 alpha/20 beta-hydroxysteroid dehydrogenase exhibits one segment sensitive to proteolysis with Glu-C protease and trypsin (cleaving after Glu192 and Arg196, respectively). Cleavage is associated with dehydrogenase inactivation; the presence of NADH offers almost complete protection and substrate (cortisone) gives some protection. Distantly related insect alcohol dehydrogenase is more resistant to proteolysis, but cleavage in a corresponding segment is detectable with Asp-N protease (cleaving before Asp198), while a second site (at Glu243) is sensitive to cleavage with both Glu-C and Asp-N proteases. Combined, the results suggest the presence of limited regions especially sensitive to proteolysis and the possibility of some association between the enzyme active site and the sensitive site(s). Modification of the hydroxysteroid dehydrogenase with tetranitromethane is paralleled by enzyme inactivation. With a 10-fold excess of reagent, labeling corresponds to 1.2 nmol Tyr/nmol protein chain and is recovered largely in Tyr152, with lesser amounts in Tyr251. Tetranitromethane also rapidly inhibits the other two dehydrogenases, but they contain Cys residues, preventing direct correlation with Tyr modification. Together, the proteolysis and chemical modifications highlight three segments of short-chain dehydrogenase subunits, one mid-chain, containing Tyr152 of the steroid dehydrogenase (similar numbers in the other enzymes), strictly conserved and apparently close to the enzyme active site, the other around position 195, sensitive to proteolysis and affected by coenzyme binding, while the third is close to the C-terminus.  (+info)

(8/27) Cooperativity between 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase in the lumen of the endoplasmic reticulum.

The functional coupling of 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase was investigated in rat liver microsomal vesicles. The activity of both enzymes was latent in intact vesicles, indicating the intraluminal localization of their active sites. Glucose-6-phosphate, a substrate for hexose-6-phosphate dehydrogenase, stimulated the cortisone reductase activity of 11beta-hydroxysteroid dehydrogenase type 1. Inhibition of glucose-6-phosphate uptake by S3483, a specific inhibitor of the microsomal glucose-6-phosphate transporter, decreased this effect. Similarly, cortisone increased the intravesicular accumulation of radioactivity upon the addition of radiolabeled glucose-6-phosphate, indicating the stimulation of hexose-6-phosphate dehydrogenase activity. A correlation was shown between glucose-6-phosphate-dependent cortisone reduction and cortisone-dependent glucose-6-phosphate oxidation. The results demonstrate a close cooperation of the enzymes based on co-localization and the mutual generation of cofactors for each other.  (+info)