Browsing by Subject "Hydroxysteroid Dehydrogenases"
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Item Influence of Cellular Localization on Activity of Hydroxysteroid Dehydrogenases(2011-12-14) Wooding, Kerry Michael; Auchus, Richard J.Hydroxysteroid dehydrogenases (HSDs) catalyze the interconversion of inactive steroids and active hormones. HSDs use nicotinamide cofactors in the cytosol and endoplasmic reticulum (ER) lumen to either reduce or oxidize their steroid substrates. Our lab has extensively studied the 17beta-HSDs types 1, 2 and 3 of the short-chain oxidoreductase family, particularly human 17beta-HSD1, which favors estrone reduction to estradiol. Rat AKR1C9 has also been thoroughly studied as a model HSD of the aldo-keto reductase family; AKR1C9 catalyzes the reduction of dihydrotestosterone to androstanediol. These two enzymes provide a basis for comparative studies with 11beta-HSD1, which catalyzes the reduction of cortisone to cortisol. Most mammalian cells supplied with adequate glucose and oxygen maintain cytoplasmic high nicotinamide concentration gradients, [NADPH] >> [NADP+] and [NAD+] >> [NADH], and in the strongly oxidizing environment of the ER lumen, both these gradients are shifted to more oxidized cofactor. Whereas 17betaHSD types 1, 2, 3 and AKR1C9 catalyze their respective reactions in a thermodynamically predictable manner based on cofactor gradients, 11beta-HSD1 does the opposite. 17beta-HSD1, 17beta-HSD3, and AKR1C9 favor reduction in the cytosol using NADPH, and 17beta-HSD2 favors oxidation in the ER lumen using NAD+. In contrast, 11beta-HSD1 reduces cortisone to cortisol in the highly oxidative ER lumen but requires hexose-6-phosphate-dehydrogenase (H6PD) to regenerate NADPH in the ER lumen. We hypothesize that H6PD directly channels NADPH to 11beta-HSD1 through specific interactions. To test this hypothesis, we have targeted 17beta-HSD1 and AKR1C9 to the ER lumen rather than the cytosol. Conversely, we have targeted 11beta-HSD1 to the cytoplasmic surface of the ER. In addition, we have engineered point mutations in 17beta-HSD1, AKR1C9, 11beta-HSD1 and H6PD, designed to attenuate the directional preferences by altering cofactor binding. Targeting and retaining 17beta-HSD1 in the ER lumen proved troublesome; regardless of the transfection conditions, ER-targeted 17beta-HSD1 was always detected in the cytosol. We conclude that either 17beta-HSD1’s activity or structure causes its translocation to the cytosol.Item Structural and Physiologic Determinants of Estrone/Estradiol Metabolism Catalyzed by Human 17beta -Hydroxysteroid Dehydrogenases Types 1 and 2(2006-07-13) Sherbet, Daniel P.; Andersson, StefanThe 17beta -hydroxysteroid dehydrogenases (17beta -HSDs) types 1 and 2 interconvert the weak and potent estrogens estrone and 17beta -estradiol. In intact cells, each enzyme exhibits a strong directional preference that favors either oxidation (17beta -HSD2) or reduction (17beta -HSD1). A positively charged arginine (R38) adjacent to the 2'-phosphate stabilizes NADP(H) binding to 17beta -HSD1 and favors reduction due to the high cytoplasmic NADPH/NADP+ ratio. In contrast, 17beta -HSD2 has a negatively charged glutamate (E116) at the position corresponding to R38 of 17beta -HSD1, which presumably repels the 2'-phosphate of NADP(H) and favors oxidation by harnessing the high cytoplasmic NAD+/NADH ratio. Substitution of a negatively charged aspartate, but not neutral glycine, for R38 of 17beta -HSD1 markedly reduces the affinity for NADP(H) and reverses the directional preference to oxidation in intact cells. We hypothesized that E116 confers oxidative preference to 17beta -HSD2 and that substitution of either a neutral or a positively charged residue for E116 would reverse the directional preference to favor reduction. Mutations E116G, E116R, and the double mutation E116G+N117R failed to attenuate the >95% oxidative preference of 17beta -HSD2 in intact cells. Affinity for all cofactors, as estimated by Km values, were measured for wild-type and mutant 17beta -HSD2 enzymes in yeast microsomes. For wild-type 17beta -HSD2, affinity for NAD(H) is nearly 1000-fold greater than for NADP(H), and the mutant enzymes retain high affinity for NAD(H) yet only slightly better affinity for NADP(H). We conclude that the directional preference of 17beta -HSD1 is principally governed by electrostatic interactions between R38 and the 2'-phosphate of NADP(H), but that the oxidative preference of 17beta -HSD2 is not solely due to E116 in the cofactor-binding domain. These data suggest that the directional preference of 17beta -HSD2 is controlled by other aspects of its cofactor-binding domain, such as the size of the cofactor-binding pocket.