In steroid biosynthesis, human dehydroepiandrosterone sulfotransferase (DHEA-ST) in the adrenals has been reported to catalyze the transfer of the sulfonate group from 3-phosphoadenosine-5-phosphosulfate to dehydroepiandrosterone (DHEA). DHEA and its sulfate play roles as steroid precursors; however, the role of the enzyme in the catabolism of androgens is poorly understood. Androsterone sulfate is clinically recognized as one of the major androgen metabolites found in urine. Here it is demonstrated that this enzyme recognizes androsterone (ADT) as a cognate substrate with similar kinetics but a 2-fold specificity and stronger substrate inhibition than DHEA. The structure of human DHEA-ST in complex with ADT has been solved at 2.7 Å resolution, confirming ADT recognition. Structural analysis has revealed the binding mode of ADT differs from that of DHEA, despite the similarity of the overall structure between the ADT and the DHEA binary complexes. Our results identify that this human enzyme is an ADT sulfotransferase as well as a DHEA sulfotransferase, implying an important role in steroid homeostasis for the adrenals and liver.Sulfonation is catalyzed by a family of sulfotransferases that conjugate a sulfonate group (SO 3 ) from 3Ј-phosphoadenosine-5Ј-phosphosulfate (PAPS) 1 to a hydroxyl group of the recipient molecule. With desulfation by sulfatases, sulfonation has been considered as one of the major enzymatic reactions in the metabolism not only of endogenous compounds and xenobiotics, but also of steroid hormones. In most cases, the transfer of the charged sulfonate moiety to an acceptor steroid decreases the biological activity of the steroid. Indeed, steroid sulfates resulting from this reaction are not capable of binding to or activating steroid receptors. In addition, the sulfonation reaction increases water solubility of steroids and thereby enhances their excretion into the urine and/or bile (1, 2).Human dehydroepiandrosterone sulfotransferase (DHEA-ST; SULT2A1; EC 2.8.2.2) was identified mainly from human liver and adrenals, using Northern blot analysis (3) and RT-PCR analysis (4). A single isoform of DHEA-ST from human liver and adrenal tissues was confirmed by the expression and purification of the enzyme from these organs (5, 6), molecular cloning studies (7) and the comparison study of the physical, kinetic, and immunological properties of liver and adrenal forms of the enzyme (8). Steroid sulfonation has been recognized as an important means for maintaining steroid hormone levels in their metabolism. In humans, dehydroepiandrosterone sulfate (DHEAS) is the most prodigious steroid precursor and one of the major secretory products of both adult and fetal adrenals. In the fetoplacental-maternal unit (the unique interdependence of fetus, placenta, and mother) shown in Scheme 1, DHEAS plays an important role as the major precursor for placental estrogen biosynthesis, thus maintaining pregnancy. A considerable amount of DHEAS is mainly produced from the fetal zone in the adrenal gland (9). Then DHEAS...

Human estrogenic 17beta-hydroxysteroid dehydrogenase is an NADP(H)-preferring enzyme. It possesses 11- and 4-fold higher specificity toward NADP(H) over NAD(H) for oxidation and reduction, respectively, as demonstrated by kinetic studies. To elucidate the roles of the amino acids involved in cofactor specificity, we generated variants by site-directed mutagenesis. The results showed that introducing a positively charged residue, lysine, at the Ser12 position increased the enzyme's preference for NADP(H) more than 20-fold. Substitution of the negatively charged residue, aspartic acid, into the Leu36 position switched the enzyme's cofactor preference from NADPH to NAD with a 220-fold change in the ratio of the specificity toward the two cofactors in the case of oxidation. This variant dramatically abolished the enzyme's reductase function and stimulated its dehydrogenase activity, as shown by enzyme activity in intact cells. The substrate-binding pocket was also studied with four variants: Ser142Gly, Ser142Cys, His221Ala, and Glu282Ala. The Ser142Gly variant abolished most of the enzyme's oxidation and reduction activities. The residual reductase activity in vitro is less than 2% that of the wild-type enzyme. However, the Ser142Cys variant was fully inactive, both as a partially purified protein and in intact cells. This suggests that the bulky sulfhydryl group of cysteine entirely disrupted the catalytic triad and that the Ser142 side chain is important for maintaining the integrity of this triad. His221 variation weakened the apparent affinity for estrone, as demonstrated by a 30-fold increase in Michaelis-Menten constant, supporting its important role in substrate binding. This residue may play an important role in substrate inhibition via the formation of a dead-end complex. The formerly suggested importance of Glu282 could not be confirmed.

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