The 20 alpha-hydroxysteroid dehydrogenase of Streptomyces hydrogenans

In addition to the well-known 3 alpha,20 beta-hydroxysteroid dehydrogenase ('cortisone reductase'), Streptomyces hydrogenans produces a relatively stable, NAD-dependent 20 alpha-hydroxysteroid dehydrogenase of molecular mass approximately 48 kDa. This enzyme catalyzes the transfer of hydrogen from the 4-pro-S position of NADH.     

17Beta-hydroxysteroid dehydrogenase (17beta-HSD) in scleractinian corals and zooxanthellae

Steroid metabolism studies have yielded evidence of 17β-hydroxysteroid dehydrogenase (17β-HSD) activity in corals. This project was undertaken to clarify whether there are multiple isoforms of 17β-HSD, whether activity levels vary seasonally, and if zooxanthellae contribute to activity. 17β-HSD activity was characterized in zooxanthellate and azooxanthellate coral fragments collected in summer and winter and in zooxanthellae cultured from Montipora capitata. More specifically, 17β-HSD activity was characterized with regard to steroid substrate and inhibitor specificity, coenzyme specificity, and Michaelis constants for estradiol (E₂) and NADP⁺. Six samples each of M. capitata and Tubastrea coccinea (three summers, three winters) were assayed with E₂ and NADP⁺. Specific activity levels (pmol/mg protein) varied 10-fold among M. capitata samples and 6-fold among T. coccinea samples. There was overlap of activity levels between summer and winter samples. NADP⁺ / NAD⁺ activity ratios varied from 1.6 to 22.2 for M. capatita, 2.3 to 3.8 for T. coccinea and 0.7 to 1.1 for zooxanthellae. Coumestrol was the most inhibitory of the steroids and phytoestrogens tested. Our data confirm that corals and zooxanthellae contain 17β-HSD and are consistent with the presence of more than one isoform of the enzyme.     

Lack of plasmids in Streptomyces hydrogenans

Abstract Wild-type cells of Streptomyces hydrogenans ATCC 19631, strain HY A₁, show a remarkable degree of genetic instability with regard to the biosynthesis of 17β-hydroxysteroid dehydrogenase. As plasmids might be responsible for this phenomenon we tried to detect plasmids in lysates of this microorganism. Streptomyces lividans , strain TK64 (pIJ916), was used as reference strain, containing a 19-kb plasmid with low abundancy. Whereas plasmid DNA could be shown in lysates of S. lividans TK64, no plasmid DNA was detectable in lysates of S. hydrogenans .     

Characterization of L-aspartate uptake by Streptomyces hydrogenans.

Multiple transport systems for L-aspartic acid exist in Steptomyces hydrogenans. The intracellular accumulation of L-aspartate against a concentration gradient was immediately inhibited by proton conductors, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone, 2,4-dinitrophenol or nigericin. Transport activity was gradually lost when inhibitors of protein synthesis were added. L-Aspartate transport had two pH optima at 6.5 and 4.5. At pH 6.5, two saturable transport components with different Km and Vmax values could be resolved by kinetic studies. A high-affinity system (system I) preferred the L-isomers of the anionic forms of aspartic and glutamic acid. At the same pH, a second, low-affinity system (system II) operated, which was presumably less specific than system I and also able to accept, at high concentrations, neutral amino acids. At pH 4.5, the Lineweaver-Burk plot revealed only a single catalytic component, with Km and Vmax values similar to those of system II. Again, in contrast to system I, this component showed high affinity for neutral amino acids. The data suggest that L-aspartic acid and L-glutamic acid are transported by this system as neutral zwitterionic molecules.     

Enzyme induction in Streptomyces hydrogenans. V. Characterization of testosterone-17 beta-dehydrogenase and its induction by steroids]

Testosterone 17beta-dehydrogenase can be enriched from Streptomyces hydrogenans. The enzyme dehydrogenizes testosterone with Km=13muM and estradiol-17beta with Km=21muM to the corresponding 17-ketoderivatives. NAD forms NADH with Km=125muM. The enzyme is strongly inhibited by androstandione and 17alpha-methyltestosterone. The Ki for 17alpha-methyltestosterone is 18muM. The enzyme activity increases with increasing pH up to alkali-mediated denaturation at about pH 10. The optimum temperature is at 45 degrees C. If Streptomyces hydrogenans is cultivated in the absence of steroids, the specific activity of testosterone 17beta-dehydrogenase in the cytosol of the microorganisms amounts to 10 mU/mg protein, and increases up to 10-fold if the cells are cultivated in the presence of certain steroids. Testosterone, alpha-dihydrotestosterone, beta-dihydrotestosterone, estradiol-17beta, and 17alpha-methyltestosterone are very effective inducers. Thus, for the first time, the ability of estradiol-17beta to induce an enzyme synthesis in a microorganism is shown. The steroid-dependent induction is inhibited by testosterone acetate and rifamycin SV. Cyproterone, however, does not decrease the testosterone-dependent enzyme induction of testosterone 17beta-dehydrogenase.     

17Beta-hydroxysteroid dehydrogenase from Cochliobolus lunatus: model structure and substrate specificity

A homology-built structural model of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus, a member of the short-chain dehydrogenase/reductase family, was worked out using the known three-dimensional structure of trihydroxynaphthalene reductase (EC 1.3.1.50) from Magnaporthe grisea as a template. Due to 61% sequence identity, the model also revealed a similar backbone trace. On the basis of qualitative thin-layer chromatography and comparative kinetic tests of the activity toward various potential steroid substrates, we conclude that androgens are more efficiently converted than estrogens. Their specific oxidoreduction predominantly occurs at the C17 position while no significant conversion at C3 and C20 was determined. Additionally, a thousand times effective inhibition by 5-methyl-(1,2,4)-triazolo[3,4-b]benzothiazole and no activity toward 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one indicate distinct specificies of 17beta-hydroxysteroid dehydrogenase from the fungus C. lunatus and trihydroxynaphthalene reductase. The results of the analysis of progress curve measurements for the forward and backward reactions are consistent with the Theorell-Chance reaction mechanism also predicted from the structural model. In accordance with these results, 4-androstene-3,17-dione was docked into the enzyme active site using molecular modeling and dynamics calculations.     

Crystallization and preliminary crystallographic study of 3 alpha, 20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans

3 alpha, 20 beta-Hydroxysteroid dehydrogenase, an NADH-dependent oxidoreductase isolated from Streptomyces hydrogenans , is a tetramer containing four subunits each of Mr 25,000. The enzyme has been crystallized by the vapor diffusion technique using either phosphate or borate buffered ammonium sulfate (pH between 6.0 and 8.7) as the precipitant. The crystals are hexagonal bipyramids ; they have the symmetry of space group P6(4)22 (or P6(2)22), with unit cell dimensions a = 127.3 A, c = 112.2 A. Volume and density considerations imply that the crystallographic asymmetric unit contains two monomers, and therefore that the tetramer possesses a 2-fold axis of symmetry that is coincident with a crystallographic 2-fold symmetry element.     

17 Beta-hydroxysteroid dehydrogenase in human breast cancer: analysis of kinetic and clinical parameters

In this study, we assessed the rate of estradiol degradation via the 17 beta-hydroxysteroid dehydrogenase (HSD) enzyme in breast tumors from postmenopausal women. We initially studied the effects of time, level of enzyme activity, amount of tissue assayed, and substrate concentration on the linearity of conversion of estradiol to estrone in breast tumor homogenates. The reaction was demonstrated to be linear when less than 15% conversion of estradiol to estrone occurred over 30 min with homogenates produced from 2.5 mg of tissue. Detailed kinetic experiments demonstrated the presence of two classes of enzyme activity, one with high affinity and the other with low affinity. In 83% of the tumors examined, the high affinity form was present and had a median Km of 0.62 microM and Vmax of 82 nmol/g protein/h. In 29 tumors, HSD activity could be precisely quantified and correlated with clinical parameters. No statistically significant correlation of enzyme activity with estrogen receptor (r2 = 0.06) or progesterone receptor (r2 = 0.006) or with patient age could be detected (r2 = 0.001). In 12 additional tumors, activity exceeded 15% conversion of estradiol to estrone at 30 min and precise quantitation was not possible. The average content of progesterone receptor was similar for these 12 tumors as for the 19 with lower HSD activity. However, estrogen receptor content and patient age were lower in the group with high HSD activity. The finding of a high affinity form of HSD in this study provides support for the biological importance of this enzyme in breast cancer tissues.     

Beta-hydroxysteroid dehydrogenase: activity in microemulsion and extraction from Pseudomonas testosteroni cells with microemulsion

The stability of purified beta-hydroxysteroid dehydrogenase activity measured as a function of time was good in buffered cationic and non-ionic microemulsions. The use of 1-pentanol and 1-hexanol in place of 1-butanol as cosurfactant gave increased activity and stability. The NAD+ Michaelis constant was 0.22 mM in buffer and 3.5 mM in waterpool concentration in microemulsion. Proteins, among them beta-hydroxysteroid dehydrogenase, were extracted from Pseudomonas testosteroni with cationic microemulsion, thus indicating that microemulsions may be utilized in protein release from cells.     

Inactivation of Streptomyces hydrogenans 20 beta-hydroxysteroid dehydrogenase by an enzyme-generated ethoxyacetylenic ketone in the presence of a thiol

Replacement of the 21-methyl group of 20 beta-hydroxypregn-4-en-3-one with an ethoxyacetylene group yields a compound that is an excellent substrate (pH 7.4, Km = 2.3 microM, Vmax = 4.6 nmol min-1 micrograms-1) for the Streptomyces hydrogenans NAD(H)-dependent 20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53). The enzyme-generated ethoxyacetylenic ketone product is a potent inactivator of the enzyme. Gel filtration chromatography of enzyme inactivated with radiolabeled steroid demonstrates that covalent modification of the enzyme has occurred. Both NAD and NADH retard the rate of inactivation, suggesting that only free enzyme is susceptible to covalent modification. Consequently, enzymatically formed ethoxyacetylenic ketone does not react with the enzyme while it is part of the ternary complex. Moreover, the kinetically preferred release of this reactive ketone prior to NADH release assures that enzyme inactivation occurs only when released ketone subsequently encounters free enzyme. Kinetic analysis of inactivations carried out with chemically prepared ethoxyacetylenic ketone and enzyme at pH 7.4 and 9.2 yields bimolecular rate constants for the inactivation process of 1.15 X 10(4) L mol-1 s-1 and 6.94 X 10(4) L mol-1 s-1, respectively. This bimolecular reaction is faster than the bimolecular reaction of the ethoxyacetylenic ketone with either glutathione, mercaptoethanol, or dithiothreitol. Thus, complete inactivation by ketone generated from 5 microM alcohol and 5 microM NAD occurs in 30 min at pH 7.4 in the presence of 1 mM glutathione.     

Inhibition of Streptomyces hydrogenans 3 alpha,20 beta-hydroxysteroid dehydrogenase by licorice-derived compounds and crystallization of an enzyme-cofactor-inhibitor complex.

Streptomyces hydrogenans 3 alpha,20 beta-hydroxysteroid dehydrogenase reduces the C20 ketone on glucocorticoids and progestins. We find that two licorice-derived compounds, glycyrrhizic acid and carbenoxolone, inhibit this enzyme with microM Kis. Inhibition is competitive, indicating that these compounds are binding at or close to the catalytic site. Carbenoxolone's high aqueous solubility and affinity for 3 alpha,20 beta-hydroxysteroid dehydrogenase enabled us to prepare crystals of a carbenoxolone-NADH-enzyme ternary complex, which preliminary X-ray analysis indicates has a crystal structure that is significantly different from that of the 3 alpha,20 beta-hydroxysteroid dehydrogenase-NADH complex. A comparison of the tertiary structures of these two complexes should prove useful in understanding this enzyme's catalytic mechanism, as well as those of two homologous enzymes, mammalian 11 beta-hydroxysteroid dehydrogenase and 15-hydroxyprostaglandin dehydrogenase that also are inhibited by carbenoxolone.     

Production of monoclonal antibodies against 3α,20β-hydroxysteroid dehydrogenase from Streptomyces hydrogenans

Abstract A procedure is described for the production of monoclonal antibodies (mAbs) against 3α,20β-hydroxysteroid dehydrogenase (3α,20β-HSD) from the actinomycete Streptomyces hydrogenans ATCC 19631. Clones which were obtained after fusion of immune cells were screened by solid-phase ELISA and immunoblotting. About 5.2% of the clones secreted immunoglobulins with specificity for 3α,20β-HSD. The purified mAbs were found to belong to subclass IgG₁ and to recognize both the native enzyme as well as its identical subunits which were obtained by SDS denaturation. However, the activity of the tetrameric holoenzyme was only weakly diminished in the presence of these mAbs.     

Regulation of amino acid transport in growing cells of Streptomyces hydrogenans

The uptake of various amino acids into Streptomyces hydrogenans grown in chemostatically and turbidostatically controlled steady state cultures has been investigated. A close correlation between transport capacity and the growth rates of the cells was found. As shown by kinetic analysis, the increased transport is due to elevated maximum uptake rates, the apparent Michaelis constants remaining unchanged. Analysis of the unidirectional fluxes of cycloleucine revealed that not only the influx is raised as the growth rate is increased but also the efflux. Hence, the conclusion is drawn that the growth-rate dependent modulation of transport capacity is, at least, partially due to the variation of the concentration of active transport components. Since the cells were grown in the absence of external amino acids the results suggest that amino acid transport into S. hydrogenans is under the control of endogenous effectors.List of Abbreviations AIB 2-aminoisobutyric acid - Cycloleucine 1-aminocyclopentane-1-carboxylic acid