Identifying Androsterone (ADT) as a Cognate Substrate for Human Dehydroepiandrosterone Sulfotransferase (DHEA-ST) Important for Steroid Homeostasis

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. 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 (SO3) from 3′-phosphoadenosine-5′-phosphosulfate (PAPS) 1The abbreviations used are: PAPS, 3′-phosphoadenosine-5′-phosphosulfate; DHEA, dehydroepiandrosterone; ADTS, androsterone sulfate; DHEAS, dehydroepiandrosterone sulfate; ADT, androsterone; DHEA-ST, dehydroepiandrosterone sulfotransferase; PAP, 3′-phosphoadenosine-5′-phosphate; EST, estrogen sulfotransferase; HSD, hydroxysteroid dehydrogenase; r.m.s., root mean-squared. 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.Strott C.A. Endocr. Rev. 1996; 17: 670-697Crossref PubMed Scopus (171) Google Scholar, 2.Falany C.N. FASEB J. 1997; 11: 206-216Crossref PubMed Scopus (530) Google Scholar). Human dehydroepiandrosterone sulfotransferase (DHEA-ST; SULT2A1; EC 2.8.2.2) was identified mainly from human liver and adrenals, using Northern blot analysis (3.Luu-The V. Dufort I. Paquet N. Reimnitz G. Labrie F. DNA Cell Biol. 1995; 14: 511-518Crossref PubMed Scopus (55) Google Scholar) and RT-PCR analysis (4.Javitt N.B. Lee Y.C. Shimizu C. Fuda H. Strott C.A. Endocrinology. 2001; 142: 2978-2984Crossref PubMed Scopus (75) Google Scholar). 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.Falany C.N. Vazquez M.E. Kalb J.M. Biochem. J. 1989; 260: 641-646Crossref PubMed Scopus (182) Google Scholar, 6.Adams J.B. McDonald D. Biochim. Biophys. Acta. 1979; 567: 144-153Crossref PubMed Scopus (48) Google Scholar), molecular cloning studies (7.Whittemore R.M. Pearce L.B. Roth J.A. Biochemistry. 1985; 24: 2477-2482Crossref PubMed Scopus (62) Google Scholar) and the comparison study of the physical, kinetic, and immunological properties of liver and adrenal forms of the enzyme (8.Comer K.A. Falany C.N. Mol. Pharmacol. 1992; 41: 645-651PubMed Google Scholar). 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.Seron-Ferre M. Lawrence C.C. Siiteri P.K. Jaffe R.B. J. Clin. Endocrinol. Metab.. 1978; 47: 603-609Crossref PubMed Scopus (101) Google Scholar). Then DHEAS is hydroxylated primarily in the fetal liver and partly in the fetal adrenal itself (10.Perez-Palacios G. Perez A.E. Jaffe R.B. J. Clin. Endocrinol. Metab.. 1968; 28: 19-25Crossref PubMed Scopus (31) Google Scholar). In the placenta, the hydroxylated DHEAS is desulfated and aromatized to form estriol, which increases uteroplacental blood flow, and then is secreted into the maternal circulation (11.Goebelsmann U. Jaffe R.B. Acta Endocrinol. (Copenh). 1971; 66: 679-693Crossref PubMed Scopus (28) Google Scholar). Androsterone sulfate (ADTS) is the most abundant circulating 5α-reduced androgen metabolite in serum (12.Zwicker H. Rittmaster R.S. J. Clin. Endocrinol. Metab. 1993; 76: 112-116Crossref PubMed Scopus (13) Google Scholar), while DHEAS is the major precursor for the active steroid hormones. A fraction of dehydroepiandrosterone (DHEA) is metabolized in liver, resulting in androstenedione and the double bond of the latter compound is reduced by 5α-reductase, giving rise to 5α- and 5β-androstanedione. The reduction of the ketone and conjugation reaction at C3 produces mainly 5α-androsterone (ADT) and etiocholanolone (or 5β-ADT), glucurono- and sulfo-conjugates, among various metabolites. More interestingly, the major portion of testosterone is oxidized to androstenedione in liver, following the same metabolism as described above (13.Milgrom E. Baulieu E-E. Kelly P.A. Steroid Hormones. Chapman and Hall, New York and London1990: 398-402Google Scholar). With other conjugation reaction, ADT sulfonation has been considered as one of the major catabolism processes of androgens in human liver before urinary excretion since a considerable amount of ADTS was identified in urine (14.De la Torre R. de la Torre X. Alia C. Segura J. Baro T. Torres-Rodriguez J.M. Anal. Biochem. 2001; 289: 116-123Crossref PubMed Scopus (53) Google Scholar). Nevertheless, it is quite interesting that a steroid sulfotransferase enzyme, other than DHEA-ST, responsible for ADT sulfonation in human liver has not been reported so far. This interested us to do a detailed kinetic study on DHEA-ST for various steroids, among which ADT was found to exert a similar activity and substrate inhibition pattern. The latter resulted in reexamination of specificity and substrate inhibition for ADT and DHEA by this enzyme. The two steroids seem quite similar viewing from the plan of the steroid core. However, when looking in detail, a DHEA molecule is stereospecifically distinct from an ADT molecule in their A rings: DHEA as a 3β-hydroxysteroid and ADT as a 3α-hydroxysteroid (Fig. 1). This stereospecific difference has intrigued us in view of the binding mode of both steroids in the substrate binding site of DHEA-ST.Fig. 1Comparison of two stereospecific substrates, DHEA and ADT. Both steroids are compared in two dimensional (A) and three-dimensional (B) structures.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Until now, two crystal structures of the enzyme have been available: SULT2A1 in complex with 3′-phosphoadenosine-5′-phosphate (PAP) (the PAP complex) (15.Pedersen L.C. Petrotchenko E.V. Negishi M. FEBS Lett. 2000; 475: 61-64Crossref PubMed Scopus (91) Google Scholar) and DHEA-ST in complex with DHEA (the DHEA complex) (16.Rehse P. Zhou M. Lin S.X. Biochem. J. 2002; 364: 165-171Crossref PubMed Scopus (70) Google Scholar). In this study, we report the crystallographic structure of the enzyme in complex with ADT, a 3α-hydroxysteroid, and describe enzyme kinetics addressing substrate specificity and substrate inhibition patterns toward DHEA and ADT. Materials—DHEA, ADT, and PAPS were obtained from Sigma Chemical Co. (9,11-3H (N))-ADT (54 Ci/mmol) and (4-14C)-DHEA (53 mCi/mmol) were purchased from PerkinElmer Life Sciences. Scintillation mixture solution, glutathione-Sepharose 4B, Q-Sepharose fast flow, and Factor Xa were from Amersham Biosciences. Purification and Sulfotransferase Assay—Preparation of homogeneous DHEA-ST was performed as described previously (17.Chang H.J. Zhou M. Lin S.X. J. Steroid Biochem. Mol. Biol. 2001; 77: 159-165Crossref PubMed Scopus (17) Google Scholar). In brief, human DHEA-ST expressed as a glutathione sulfotransferase fusion form from Escherichia coli was purified using glutathione-Sepharose 4B affinity chromatography, a Factor Xa cleavage step, and Q Sepharose anion exchange column chromatography. Purified protein was confirmed by SDS-PAGE and stored at –20 °C with 50% glycerol before using. No big change for the sulfating activity was observed during storage. DHEA-ST activity assay was performed as mentioned previously (17.Chang H.J. Zhou M. Lin S.X. J. Steroid Biochem. Mol. Biol. 2001; 77: 159-165Crossref PubMed Scopus (17) Google Scholar) with little modification. DHEA-ST activity was assayed at 37 °C at various time intervals in a final reaction volume of 150 μl containing 20 mm Tris, pH 7.5, 15 mm MgCl2, 50 μm PAPS, 2% ethanol, and various amounts of steroids. The reaction was stopped by adding the equivalent volume of xylene, vortexing, and centrifuging for 10 min at 3000 rpm to divide into the aqueous and the solvent phases. The phases were completely separated with ethanol-dry ice bath. Each phase of 80 μl was used to determine the amount of sulfate-conjugated steroids by liquid scintillation counting in a Beckman LS 3801 (Irvine, CA). One unit of enzyme is defined as the amount of enzyme that catalyzes the formation of 1 nmol of each steroid sulfate per min under the conditions mentioned above. Kinetic Studies and Data Processing—All reactions were performed at 37 °C and pH 7.5 using 0.1–6.25 μg of enzyme, a wide range of the steroid concentrations (0.05–40 μm) and a saturating concentration of the cofactor, PAPS, (50 μm) in the reaction mixture. The initial velocities were measured with less than 10% substrate conjugation. For the determination of all the kinetic constants, at least 2–3 independent experiments were performed and then the mean value was taken. Initial velocity data in the range of non-inhibitory substrate concentrations (for DHEA and ADT) were first individually fitted to the Michaelis-Menten Equation 1. Michaelis constant (Km) and Maximal velocity (Vmax) for all the steroids examined were calculated using the corresponding double-reciprocal plots (Equation 2). Data for substrate inhibition were fitted to substrate inhibition in Equation 3 (18.Cornish-Bowden A. Analysis of Enzyme Kinetic Data. Oxford University Press, Oxford1995: 118-122Google Scholar). The real maximal velocity (Vmax) and the substrate concentration at that velocity were calculated by Equations 4 and 5 that were derived mathematically from Equation 3 (19.Gangloff A. Garneau A. Huang Y.W. Lin S.X. Biochem. J. 2001; 356: 269-276Crossref PubMed Google Scholar). v=V[S]/(Km+[S])(Eq. 1) 1/v=Km[S]V+1/V(Eq. 2) v=V[S]/{Km+[S](1+[S]/Kis)}(Eq. 3) Vmax=V/{1+2√(Km/Kis)}(Eq. 4) s=√(KmKis)(Eq. 5) v is the experimentally determined initial velocity, V is the maximal velocity, [S] is the concentration of the variable substrate, Km is the concentration of substrate at half-maximal velocity, Kis is the substrate inhibition constant, Vmax is the real maximal velocity calculated theoretically, and s is the substrate concentration at which the real maximal velocity is reached. The molecular mass of the monomer used to calculate the kcat value was 34 kDa (5.Falany C.N. Vazquez M.E. Kalb J.M. Biochem. J. 1989; 260: 641-646Crossref PubMed Scopus (182) Google Scholar, 17.Chang H.J. Zhou M. Lin S.X. J. Steroid Biochem. Mol. Biol. 2001; 77: 159-165Crossref PubMed Scopus (17) Google Scholar). Crystallization and Data Collection—DHEA-STs were co-crystallized with ADT using the hanging drop vapor diffusion technique at room temperature (20.Zhou M. Rehse P. Chang H.J. Luu-The V. Lin S.X. Acta Crystallogr. D. Biol. Crystallogr. Sect. D. 2001; 57: 1630-1633Crossref PubMed Scopus (9) Google Scholar). Proteins were concentrated at 15 mg/ml in 10 mm Tris, pH 7.5, 0.1% n-octyl β-d-glucopyranoside (β-OG). Drops were prepared by mixing equal volumes of protein and reservoir solutions consisting of 1.6 m ammonium sulfate, 0.1 m HEPES, pH 7.5, and 0.1 m sodium chloride. Then 0.27 μlof10mm ADT was added to the drop. The crystals appeared after 2–3 days and matured in 7–10 days. Diffraction data were collected from one cryo-cooled crystal at beamline X8C at the National Synchrotron Light Source, Brookhaven National Laboratory while the crystal was oscillated through 1o steps. The ADT complex crystal belongs to orthorhombic space group P21212 with unit cell dimensions of a = 75.3 Å, b = 129.8 Å, c = 44.3 Å. The data were processed using HKL suite (21.Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38773) Google Scholar). Structure Determination and Model Refinement—Structure solution was determined by molecular replacement using the CNS software package (22.Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D. Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (17024) Google Scholar). We used the refined 2.5 Å resolution structure of the human DHEA-ST in complex with DHEA (accession code 1J99 in Protein Data Bank) as the starting model. The initial electron density maps were calculated using 2Fo – Fc and Fo – Fc coefficients (where Fo and Fc are the observed and calculated structure factors respectively). The model was updated until completion using the CNS software package for refinement (22.Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D. Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (17024) Google Scholar) and the O program for model building (23.Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A Fund. Crystallogr. 1991; 47: 110-119Crossref PubMed Scopus (13036) Google Scholar). After the structure was rebuilt and refined by several cycles, the electron density corresponding to ADT and sulfate group was clear enough to put the two molecules in the substrate binding site and the cofactor-binding site respectively. The stereochemistry of the final model was verified with the program PROCHECK (24.Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Details of the data collection and refinement statistics for the model are given in Table I. Coordinates have been deposited in the Protein Data Bank (25.Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (28971) Google Scholar) with the accession number 1OV4. Structural comparison studies using the ADT complex, the DHEA complex, the PAP complex (accession code 1EFH in Protein Data Bank), and the estrogen sulfotransferase (EST) structure (accession code 1AQU in Protein Data Bank) were performed using the lsq routines of the O program (23.Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A Fund. Crystallogr. 1991; 47: 110-119Crossref PubMed Scopus (13036) Google Scholar).Table ISteady-state kinetics for the sulfonation reaction of DHEA-ST with ADT and DHEASteroidKmVmaxkcatKisSpecificityμmU/mgs-1μmμm-1 s-1ADT2.1 ± 0.5221.1 ± 36.40.13 ± 0.023.8 ± 0.90.062DHEA3.1 ± 0.7180.3 ± 27.10.1 ± 0.01510.6 ± 2.40.032 Open table in a new tab Steady-state Kinetics and Substrate Inhibition for DHEA and ADT—The steady-state kinetics for DHEA and ADT were studied as described under “Experimental Procedures.” The initial velocity versus steroid concentration plots and the corresponding Lineweaver-Burk plots are shown in Fig. 2, A and B for DHEA and ADT, respectively. In the inserts of Fig. 2, the initial velocity increases with substrate concentrations up to 2 μm for ADT and 4–6 μm for DHEA and then decreases with increasing substrate concentrations, identifying a substrate inhibition pattern. At very high substrate concentrations, this velocity reaches a minimum level of 30 units/mg for ADT and 40 units/mg for DHEA, rather than falling to zero. As depicted in Fig. 2, the Lineweaver-Burk plots are by no means linear compared with those of general Michaelis-Menten kinetics. Three regions of the plots can generally be discerned: the region that follows Michaelis-Menten kinetics, the transition region, and the substrate inhibition region. At low concentrations of DHEA (2 μm and lower) and ADT (1 μm and lower), the double-reciprocal plot gives a linear range that follows the Michaelis-Menten equation. The sulfonation reaction was largely inhibited when higher concentrations of DHEA (20 μm and higher) and ADT (10 μm and higher) were used and 1/v value increased rapidly following the decrease of 1/S value (the increase of substrate concentration) in this range. A transition phase of 1–10 μm for ADT and 2–20 μm for DHEA showed the transfer from the Michaelis-Menten zone to the substrate inhibition area. The kinetic constants were calculated when the whole range of experimental data was analyzed by the substrate inhibition equation v = V × [S]/{Km + [S](1+[S]/Kis)} (Table II). We can see that the enzyme catalyzes the transfer of sulfonate group from PAPS toward ADT with a Km = 2.1 ± 0.5 μm and kcat = 0.13 ± 0.02 s–1 while the kinetic constants for the DHEA molecule are Km = 3.1 ± 0.7 μm and kcat = 0.1 ± 0.015 s–1. The specificity (kcat/Km) of ADT is twice that of DHEA as shown in Table II, indicating that ADT is at least as specific as DHEA for the enzyme. Moreover at a certain high concentration range of ADT (Kis = 3.8 ± 0.9 μm), substrate inhibition is induced as in the case of DHEA (Kis = 10.6 ± 2.4 μm), which is a unique feature in the sulfotransferase family (26.Otterness D.M. Wieben E.D. Wood T.C. Waston R.W.G. Madden B.J. McCormick D.J. Weinshiboum R.M. Mol. Pharmacol. 1992; 41: 865-872PubMed Google Scholar, 27.Sugiyama Y. Stolz A. Sugimoto M. Kuhlenkamp J. Yamada T. Kaplowitz N. Biochem. J. 1984; 224: 947-953Crossref PubMed Scopus (11) Google Scholar, 28.Marcus C.J. Sekura R.D. Jakoby W.B. Anal. Biochem. 1980; 107: 296-304Crossref PubMed Scopus (53) Google Scholar). In comparison to DHEA sulfonation, ADT is now identified as a cognate substrate to which the enzyme shows even somewhat higher specificity and stronger substrate inhibition, as presented in this work. Maximal velocities of 180.3 ± 27.1 units/mg for DHEA and 221.1 ± 36.4 units/mg for ADT, which correspond to the kcat values for the Michaelis-Menten reaction, cannot be reached directly in the experiment owing to substrate inhibition. In reality, experimental maximal velocity for the whole reaction was around 90 units/mg for both steroids before substrate inhibition begins as shown in Fig. 2. These values are very consistent with the theoretical values of 88.4 units/mg for ADT and 90.2 units/mg for DHEA obtained using the Equation 4. The substrate concentration at the real maximum velocity was also determined to be 2.82 μm for ADT and 5.73 μm for DHEA using Equation 5 mentioned under “Experimental Procedures.” These values are in good agreement with the observed substrate concentration at the maximum point for the curve drawn in Fig. 2.Table IIData collection, structural and refinement statisticsParametersValuesUnit cell dimensionsa, b, c (Å)75.29, 129.82, 44.29Space groupP21212Number of reflections36,310 (3571)Unique reflections11,911 (1189)Resolution (Å)20-2.70 (2.80-2.70)Completeness (%)95.2 (96.7)I/σ (1)11.3 (2.1)Rmerge*0.075 (0.424)Multiplicity3.1 (3.0)R-factor23.0R-free27.1Rms deviations from target geometryBond lengths (Å)0.007Bond angles (o)1.3Mean B factors and number of non-hydrogen atomsAll atoms58.14/2188Protein58.05/2137Sulfate71.40/5ADT73.19/21Water50.96/25 Open table in a new tab Overall Structure—The crystal structures of human DHEA-ST have been reported in the presence of DHEA (16.Rehse P. Zhou M. Lin S.X. Biochem. J. 2002; 364: 165-171Crossref PubMed Scopus (70) Google Scholar) and PAP (15.Pedersen L.C. Petrotchenko E.V. Negishi M. FEBS Lett. 2000; 475: 61-64Crossref PubMed Scopus (91) Google Scholar). Here the ADT binary complex structure is determined. The overall structure of the enzyme includes an ADT molecule and a modeled PAP, showing both substrate and expected cofactor binding sites (Fig. 3). The overall structure of the ADT complex is very similar to that of the DHEA complex except for some residues and the flexible loops. The root mean square deviation value between the two structures is 0.935 for the α-carbon of 267 amino acids excluding the two flexible loops formed by residues Asn-226 to Asp-237 and Leu-246 to Val-250. All data between 20 and 2.70 Å were used in the refinement, yielding a crystallographic R-factor of 23.0% and a free R-factor of 27.0%. There is one monomer in the asymmetric unit even though the active protein is a homodimer in solution (5.Falany C.N. Vazquez M.E. Kalb J.M. Biochem. J. 1989; 260: 641-646Crossref PubMed Scopus (182) Google Scholar, 17.Chang H.J. Zhou M. Lin S.X. J. Steroid Biochem. Mol. Biol. 2001; 77: 159-165Crossref PubMed Scopus (17) Google Scholar). The main core of the ADT complex structure is composed of an α/β-fold with a central four-stranded parallel β-sheet surrounded by α-helices on both sides as described in a previous report (15.Pedersen L.C. Petrotchenko E.V. Negishi M. FEBS Lett. 2000; 475: 61-64Crossref PubMed Scopus (91) Google Scholar). The refined model includes one DHEA-ST monomer, an ADT molecule and a sulfate molecule. In addition, the model includes 25 water molecules. Out of the 284 amino acids contained in the protein, 267 were modeled into the electron density in the present structure while 284 amino acids were built in the DHEA complex structure. The missing amino acids belong to loop regions that could not be built in the ADT complex structure. The Ramachandran plot showed that all residues are in allowed regions with 87.2% in the most favored regions. Substrate Binding Site—The present ADT binary complex structure is compared with both the PAP-DHEA-ST structure (the PAP binary complex structure) and the DHEA binary complex structure. The 2Fo – Fc electron density map contoured at 1 σ level enables the unambiguous localization of an ADT molecule (Fig. 4) and a sulfate group (Fig. 6) bound to DHEA-ST in the substrate binding and the cofactor binding sites respectively. The ADT complex structure has only one substrate orientation in the active site while two distinct orientations, the catalytic and the alternative, have been proposed in the DHEA complex structure (Fig. 5) (16.Rehse P. Zhou M. Lin S.X. Biochem. J. 2002; 364: 165-171Crossref PubMed Scopus (70) Google Scholar). Superimposing the two structures shows that an ADT molecule as a whole is close to the location of the DHEA molecule in the proposed alternative orientation. In the ADT complex structure, no electron density for the proposed catalytic orientation has been found since the Cϵ atom of Met-137 residue is inward the substrate binding site and results in steric hindrance against the proposed catalytic location of the DHEA molecule (Fig. 5). The active site for the sulfonation reaction is identified through the position of O-3 of ADT, which functions as a sulfonate acceptor (Fig. 5). There is little displacement of O-3 atom between the DHEA complex and the ADT complex structures (1.04 Å for the distance between O-3 of ADT and O-3 of DHEA in the alternative orientation, 1.74 Å between O-3 of ADT and O-3 of DHEA in the catalytic orientation). A hydrogen bond (3.0 Å) between O-3 of ADT and Nϵ-2 of His-99 in DHEA-ST is identified similar to that between DHEA and His-99 in the DHEA complex structure (Fig. 5). This histidine is strongly conserved among several sulfotransferase families, including EST, phenol sulfotransferase, and flavonol 3-sulfotransferase, suggesting its catalytic role (15.Pedersen L.C. Petrotchenko E.V. Negishi M. FEBS Lett. 2000; 475: 61-64Crossref PubMed Scopus (91) Google Scholar). Until now, the ADT complex and the DHEA complex structures are quite similar. If so, what is the main difference between the two structures in the catalytic center of DHEA-ST? As shown in Fig. 6, the main difference between the two structures is the steroid orientation: the ADT molecule is flipped over 180° around its long axis, in comparison to the DHEA molecule of the alternative orientation. Several factors have been involved in the flip-flop of the ADT molecule. Primarily, this takes place in order to favor the making of a hydrogen bond between O-3 of ADT and Nϵ-2 of His-99 of the enzyme. If the ADT molecule is in the same plane as the DHEA molecule considering the orientation of C-18 and C-19 atoms, O-3 of ADT (α-position) would be far away from Nϵ-2 atom with the distance of 4.6 Å due to the stereospecific difference of A ring. Therefore the hydrogen bond between O-3 of ADT and Nϵ-2 of His-99 of DHEA-ST in the ADT complex structure cannot be made. The present crystallographic data indicate that human DHEA-ST does not provide stereospecific discrimination between an O-3 α steroid (ADT) and an O-3 β steroid (DHEA), thereby supporting our kinetic results (Table II). On the contrary, guinea pig DHEA-STs showed stereospecificity, based on the comparative enzymatic study with DHEA and ADT (29.Driscoll W.J. Martin B.M. Chen H.C. Strott C.A. J. Biol. Chem. 1993; 268: 23496-23503Abstract Full Text PDF PubMed Google Scholar, 30.Park B.C. Lee Y.C. Strott C.A. J. Biol. Chem. 1999; 274: 21562-21568Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). Secondly, O-17 of ADT establishes another hydrogen bond (3.28 Å) with the hydroxyl group of Ser-80 that stabilizes the ADT molecule in this position (Fig. 5). No hydrogen bond for the O-17 atom of DHEA has been found in the DHEA complex structure. Third, several hydrophobic residues are found in the vicinity of the substrate binding site (within 6 Å). Among them, the Phe-133, Trp-134, Phe-18, and Trp-72 residues are involved in van der Waals interactions with the ADT molecule. Fourth, the side chain of Trp-77 is juxtaposed with ADT at a distance of around 4 Å, indicating that this residue provides another important interaction for ADT orientation (Fig. 6). The A and B rings of the ADT molecule are sandwiched between the side chain of residue Trp-77 on one side and the side chains of residues Phe-133 and Trp-134 on the other. This sandwich conformation makes the orientation of the A ring of ADT stable. All these observations suggest that ADT binds to DHEA-ST at least as tightly as DHEA, as shown by the apparent affinity for ADT (Km = 2.1 ± 0.5 μm) and DHEA (Km = 3.1 ± 0.7 μm) (Table II).Fig. 6Comparison of the binding mode of two 3-hydroxysteroids, DHEA, and ADT. Comparison of the binding mode was performed between the DHEA complex (yellow) and the ADT complex structures (red). The ADT molecule (magenta) is flipped over along the long axis of the steroid when compared with the DHEA molecule (green). The sulfate group introduced by the crystallization is in blue. This image was produced using O/OPLOT and Molray.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Comparison of substrate binding sites. Stereoview of the active site residues of the DHEA complex (yellow) and the ADT complex (red). The DHEA molecule placed in two proposed orientations, catalytic and alternative, is colored in green and the ADT molecule in magenta. The sulfate group introduced by the crystallization is in blue. This image was produced using O/OPLOT and Molray (36.Harris M. Jones T.A. Acta Crystallogr. D Biol. Crystallogr. 2001; 57: 1201-1203Crossref PubMed Scopus (129) Google Scholar).View Large Image