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Originally published In Press as doi:10.1074/jbc.M310446200 on October 21, 2003

J. Biol. Chem., Vol. 279, Issue 4, 2689-2696, January 23, 2004
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Identifying Androsterone (ADT) as a Cognate Substrate for Human Dehydroepiandrosterone Sulfotransferase (DHEA-ST) Important for Steroid Homeostasis

STRUCTURE OF THE ENZYME-ADT COMPLEX*

Ho-Jin Chang{ddagger}, Rong Shi{ddagger}, Peter Rehse{ddagger}§, and Sheng-Xiang Lin{ddagger}

From the {ddagger}Canadian Institutes of Health Research Group in Oncology and Molecular Endocrinology Laboratory, CHUL Research Center and Laval University, Sainte-Foy, Quebec G1V 4G2, Canada

Received for publication, September 22, 2003 , and in revised form, October 18, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Sulfonation is catalyzed by a family of sulfotransferases that conjugate a sulfonate group (SO3) from 3'-phosphoadenosine-5'-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 [EC] ) 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 is hydroxylated primarily in the fetal liver and partly in the fetal adrenal itself (10). 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). Androsterone sulfate (ADTS) is the most abundant circulating 5{alpha}-reduced androgen metabolite in serum (12), 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 5{alpha}-reductase, giving rise to 5{alpha}- and 5{beta}-androstanedione. The reduction of the ketone and conjugation reaction at C3 produces mainly 5{alpha}-androsterone (ADT) and etiocholanolone (or 5{beta}-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). 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). 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 3{beta}-hydroxysteroid and ADT as a 3{alpha}-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.



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  		SCHEME 1.
The fetoplacental-maternal unit (the unique interdependence of fetus, placenta, and mother).

 



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  		FIG. 1.
Comparison of two stereospecific substrates, DHEA and ADT. Both steroids are compared in two dimensional (A) and three-dimensional (B) structures.

 
Until now, two crystal structures of the enzyme have been available: SULT2A1 in complex with 3'-phosphoadenosine-5'-phosphate (PAP) (the PAP complex) (15) and DHEA-ST in complex with DHEA (the DHEA complex) (16).

In this study, we report the crystallographic structure of the enzyme in complex with ADT, a 3{alpha}-hydroxysteroid, and describe enzyme kinetics addressing substrate specificity and substrate inhibition patterns toward DHEA and ADT.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
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). 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) 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). 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).

(Eq. 1)

(Eq. 2)

(Eq. 3)

(Eq. 4)

(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, 17).

Crystallization and Data Collection—DHEA-STs were co-crystallized with ADT using the hanging drop vapor diffusion technique at room temperature (20). Proteins were concentrated at 15 mg/ml in 10 mM Tris, pH 7.5, 0.1% n-octyl {beta}-D-glucopyranoside ({beta}-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).

Structure Determination and Model Refinement—Structure solution was determined by molecular replacement using the CNS software package (22). We used the refined 2.5 Å resolution structure of the human DHEA-ST in complex with DHEA (accession code 1J99 [PDB] 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) and the O program for model building (23). 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). 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) with the accession number 1OV4. Structural comparison studies using the ADT complex, the DHEA complex, the PAP complex (accession code 1EFH [PDB] in Protein Data Bank), and the estrogen sulfotransferase (EST) structure (accession code 1AQU [PDB] in Protein Data Bank) were performed using the lsq routines of the O program (23).


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  		TABLE I
Steady-state kinetics for the sulfonation reaction of DHEA-ST with ADT and DHEA

Steroid sulfonation was determined using concentrations of both steroids varying from 0.05 µM to 40 µM. The reaction mixture contained 20 mM Tris/HCl, pH 7.5, 15 mM MgCl2, 50 µM PAPS, 2% ethanol, and the steroid at 37°C. The kinetic parameters were obtained using the substrate inhibition equation v = V[S]/{Km + [S](1 + [S]/Kis)}. The value of specificity was calculated as kcat/Km.

 

   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
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 x [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 (26–28). 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.



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  		FIG. 2.
Lineweaver-Burk plot for steroid sulfonation with DHEA (A) and ADT (B) as the substrate. Steroid sulfonation was determined using concentrations of both steroids varying from 0.05–40 µM. The concave shape of the curve no longer reflects standard Michaelis-Menten kinetics but shows substrate inhibition at higher concentrations of the steroids (DHEA above 4 µM and ADT 2 µM). The inset shows the v versus s plot for the same reaction. Each point was determined from at least duplicate assay data.

 


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  		TABLE II
Data collection, structural and refinement statistics

Data statistics for the last shell are given in parentheses.

 
Overall Structure—The crystal structures of human DHEA-ST have been reported in the presence of DHEA (16) and PAP (15). 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 {alpha}-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, 17). The main core of the ADT complex structure is composed of an {alpha}/{beta}-fold with a central four-stranded parallel {beta}-sheet surrounded by {alpha}-helices on both sides as described in a previous report (15). 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.



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  		FIG. 3.
The overall structure of DHEA-ST. ADT is colored in magenta and modeled PAP in cyan, showing both substrate and expected cofactor binding sites. This image was produced using SETOR (35)

 
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 {sigma} 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). 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 C{epsilon} 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 N{epsilon}-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). 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 N{epsilon}-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 ({alpha}-position) would be far away from N{epsilon}-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 N{epsilon}-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 {alpha} steroid (ADT) and an O-3 {beta} 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, 30). 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).



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  		FIG. 4.
Electron density map corresponding to the ADT bound in the enzyme's active site. Shown is a face and rotated 90° view from the 2.7 Å 2Fo – Fc electron density map contoured at 1 {sigma} level. The figure was drawn using O/OPLOT.

 



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  		FIG. 6.
Comparison 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.

 



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  		FIG. 5.
Comparison 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).

 
The loop comprising the residues Asn-226 to Asp-237 is missing in the ADT complex structure while in the DHEA complex structure this loop is situated near the substrate binding site and plays a role as a roof for this site. In the PAP complex structure, the substrate binding site is occupied by the loop (Asn-226 to Asp-237) and the aromatic ring of Trp-77. Therefore, the site is more compact in the absence of a substrate. The active site extends to a proper size to accommodate the substrate when a steroid approaches the site. This is in agreement with the recent results in the study of other steroid-converting enzymes, such as the significant active site volume modification in binding different steroids found in the type 5 human 17{beta}-hydroxysteroid dehydrogenase (HSD),2 and the conformational rearrangements found when estradiol advances in the binding site of human estrogenic 17{beta}-HSD (31).

The catalytic orientation of the DHEA molecule of the DHEA complex structure was then chosen (16) based on the orientation of PAP within the DHEA-ST molecule (15). However, the true ternary complex of the enzyme with DHEA and PAP has not been obtained, thus the proposed catalytic orientation has not yet been conclusively elucidated and requires further study and evaluation.

Cofactor Binding Site: A Gate Structure Implied in Substrate Inhibition—All sulfotransferases use only one cofactor, PAPS, as a sulfonate donor. This may explain why the cofactor binding sites of the sulfotransferase structures are more conserved than the substrate binding sites throughout the sulfotransferase families. Our substrate binary structures (the ADT complex and the DHEA complex) are compared with the PAP containing structures. As shown in Fig. 7, the main difference in the cofactor binding site is the loop (Lys-242 to Asp-253) which is explicitly open in the DHEA complex structure, whereas it is closed in the PAP complex structure. In detail, the open entrance in the DHEA complex structure has two sides with a width of around 8.4 Å: Lys-188, Phe-220, and Lys-224 on one side (side A) and Leu-246 to Gly-252 on the other side (side B) (Fig. 7). Since the electron density of this loop is not clear in the ADT complex structure, seven residues of this loop (twelve residues) have been built on the open position. Accordingly the loop in the ADT complex structure is expected to be in the open position as in the DHEA complex structure. Residues Thr-243 and Lys-242 of side A play a role as a hinge and so does residue Asp-253 in our binary complex structures. However in the PAP complex structure, side A is in the closed position on the entrance, functioning like a gate for the cofactor's entrance, while there is only little displacement on side B to accommodate the cofactor molecule as compared with our binary complex structures (Fig. 7). This gate produces additional interactions between the enzyme and PAP: a van der Waals contact with Leu-246, hydrogen bonds with Arg-247, Lys-248, and Gly-249 and a hydrophobic interaction with Leu-245. These interactions and the closed conformation are also identified in PAP containing EST structure by our structural comparison studies. The gate structure indicates that the cofactor binds more tightly than a substrate, shown by the comparison of important interactions with the enzyme (Table III). This is in agreement with the fact that the Km value for PAPS has been determined around 0.8 µM when DHEA was used as a substrate at a fixed concentration3 and those for ADT and DHEA were 2.1 ± 0.5 µM and 3.1 ± 0.7 µM, respectively. This gate structure may further explain the substrate inhibition pattern of DHEA-ST. Generally the sulfonation reaction starts by taking the sulfonate group from PAPS and transferring it to a 3-hydroxyl substrate such as ADT or DHEA. The sulfated product may be released first due to the strong association of the cofactor to the enzyme. This agrees with the fact that the dissociation constant for PAP in EST is 30 ± 0.3 nM whereas that for estradiol sulfate is 270 ± 50 nM (32). Then the other reaction product, PAP, can be freed from the enzyme into the environment before a second substrate comes to the active site for next cycle of the enzyme reaction. However, at a certain high substrate concentration, the second non-sulfated substrate binds to the active site before PAP leaves, forming a dead end complex (a nonproductive enzyme-PAP-substrate complex). The formation of the dead end complex was proposed as non-reactive PAP possessing a Kis of 0.07 µM against phenol sulfotransferase (7).



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  		FIG. 7.
A gate structure for the cofactor-binding site. The cofactor binding sites of the DHEA complex (left) and the PAP complex (right) are colored in green. The loop from Gln-244 to Asp-253 plays a role as a gate and is drawn in ribbon form. In the PAP complex structure, the same residues (side A) move into side B to close the entrance; PAP is in purple. This image was produced using O/OPLOT and Molray.

 


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  		TABLE III
Comparison of the important interactions between the enzyme and a ligand

The number of important interactions between the enzyme and PAP, ADT, or DHEA was determined from the PAP complex, the ADT complex, and the DHEA complex structures respectively, using Ligplot (37).

 

There is one sulfate group in the cofactor-binding pocket in the present ADT complex structure (Fig. 6). The sulfate group originated from the crystallization conditions (1.6 M ammonium sulfate, 0.1 M HEPES, pH 7.5, 0.1 M sodium chloride) for the ADT binary complex as described in the materials and methods section. The sulfate group is supposed to be next to the 3'-phosphate group (P-2) of PAP, if PAPS structure is considered based on the PAP complex structure. But in the ADT complex structure, the sulfate is located in a similar position to the 3'-phosphate group of PAP. Even though it is misplaced in the position corresponding to 3'-phosphate group of PAP, the present location might thermodynamically be the best for the sulfate group.

ADT Sulfotransferase in Liver as Well as DHEA Sulfotransferase in Adrenal?—No detailed study addressing ADT sulfonation by human DHEA-ST has been reported. In former reports, ADT has not been considered as a cognate substrate for human DHEA-ST due to its lower reactivity (30–70%) when compared with DHEA sulfonation (1, 11). Let us review the reactivity of ADT again from the perspective of enzyme kinetics. The ADT concentration used for investigating the relative activity was 6–10 µM, which is significantly higher than the Kis value of ADT (Kis = 3.8 ± 0.9 µM) whereas the DHEA concentration used for the same experiments was 3 µM, which is much lower than the Kis level of DHEA (Kis = 10.6 ± 2.4 µM). Therefore substrate inhibition occurs at these concentrations of ADT as shown in Fig. 2B. Accordingly, suffice it to say that the maximum velocity for ADT could be similar to or even higher than that for DHEA if the substrate inhibition is considered. This has been confirmed by our kinetic data (Km = 2.1 ± 0.5 µM, kcat = 0.13 ± 0.02 s–1 and specificity = 0.062 µM–1 s–1 for ADT and Km = 3.1 ± 0.7 µM, kcat = 0.1 ± 0.015 s–1 and specificity = 0.032 µM–1 s–1 for DHEA), indicating that ADT is at least as specific as DHEA. On top of that, human embryonic kidney (HEK)-293 cells transfected with human DHEA-ST cDNA showed sulfonation reaction toward ADT, implying that ADT sulfonation by DHEA-ST happens in cells (33).

Based on the measurements of steroid level and correlations among steroids in serum, the two sulfated steroids, ADTS and DHEAS were significantly correlated (r = 0.59) (12). This is even more interesting if we consider DHEAS the major androgen precursor and ADTS one of the main androgen metabolites in view of steroid homeostasis.

Substrate inhibition is induced at a certain high concentration range of ADT and DHEA, which is a typical phenomenon in the sulfotransferase family (26–28). The Kis level of DHEA molecule (Kis = 10.6 ± 2.4 µM) is quite high compared with the actual DHEA concentration (at most 0.79 µM) in the adrenal organ (34). This implies that substrate inhibition is not related to the physiological significance of the metabolism of steroids in human adrenals. However the substrate inhibition of ADT sulfonation (Kis = 3.8 ± 0.9 µM) produces a totally different story from a physiological point of view. The concentrations of ADT and etiocholanolone (at both sulfonated and glucuronidated form) in urine have been reported to be 6 and 2.5 µM, respectively (14). The ADT concentration in human liver might be higher than 6 µM if a precursor-to-product relationship is considered. Taking this into consideration, substrate inhibition of ADT sulfonation may control the effect of an increase of ADT levels even though there is no direct information for the levels of ADT in human liver. In steroid catabolism, this enzyme can contribute to the maintenance of the steroid level through substrate inhibition mechanism while no such controlling mechanism in producing DHEAS has been found caused by low DHEA concentration in human adrenal.

In conclusion, with glucuronidation by UDP-glucuronosyltransferases, DHEA-ST plays a pivotal role in sulfonating and metabolizing ADT in human liver. We have now demonstrated that the former reported human DHEA-ST is the ADT sulfotransferase in human liver as well as the DHEA sulfotransferase in human adrenal. Substrate inhibition of this enzyme plays a major role in maintaining the level of steroid hormones, especially those of androgens. At the same time, our study shows that substrate inhibition is useful in identifying cognate substrates for enzymes.


   FOOTNOTES
 
* This work has been supported by the Canadian Space Agency and the Canadian Institutes of Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1OV4) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). Back

§ Present address: RIKEN Harima Institute at Spring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan. Back

To whom correspondence should be addressed: CIHR Group in Oncology and Molecular Endocrinology Laboratory, CHUL Research Center and Laval University, 2705 Laurier Blvd, Sainte-Foy, Quebec G1V 4G2, Canada. Tel.: 418-654-2296; Fax: 418-654-2729; E-mail: sxlin{at}crchul.ulaval.ca.

1 The 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. Back

2 W. Qiu and S.-X. Lin, unpublished data. Back

3 H-J. Chang, Y. W. Huang, and S.-X. Lin manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Dr. F. Labrie for his interests in this work. We also wish to acknowledge Dr. M. Steel for the editing of the manuscript. Data collection was carried out at the National Synchrotron Light Source, Brookhaven National Laboratory, supported by a joint grant from the Natural Science and Engineering Council of Canada and Canadian Institutes of Health Research for a consortium for operation of a protein crystallographic synchrotron beamline.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Strott, C. A. (1996) Endocr. Rev. 17, 670–697[Abstract/Free Full Text]
  2. Falany, C. N. (1997) FASEB J. 11, 206–216[Abstract]
  3. Luu-The, V., Dufort, I., Paquet, N., Reimnitz, G., and Labrie, F. (1995) DNA Cell Biol. 14, 511–518[Medline] [Order article via Infotrieve]
  4. Javitt, N. B., Lee, Y. C., Shimizu, C., Fuda, H., and Strott, C. A. (2001) Endocrinology 142, 2978–2984[Abstract/Free Full Text]
  5. Falany, C. N., Vazquez, M. E., and Kalb, J. M. (1989) Biochem. J. 260, 641–646[Medline] [Order article via Infotrieve]
  6. Adams, J. B., and McDonald, D. (1979) Biochim. Biophys. Acta 567, 144–153[Medline] [Order article via Infotrieve]
  7. Whittemore, R. M., Pearce, L. B., and Roth, J. A. (1985) Biochemistry 24, 2477–2482[CrossRef][Medline] [Order article via Infotrieve]
  8. Comer, K. A., and Falany, C. N. (1992) Mol. Pharmacol. 41, 645–651[Abstract]
  9. Seron-Ferre, M., Lawrence, C. C., Siiteri, P. K., and Jaffe, R. B. (1978) J. Clin. Endocrinol. Metab. 47, 603–609[Abstract/Free Full Text]
  10. Perez-Palacios, G., Perez, A. E., and Jaffe, R. B. (1968) J. Clin. Endocrinol. Metab. 28, 19–25[Abstract/Free Full Text]
  11. Goebelsmann, U., and Jaffe, R. B. (1971) Acta Endocrinol. (Copenh). 66, 679–693[Abstract/Free Full Text]
  12. Zwicker, H., and Rittmaster, R. S. (1993) J. Clin. Endocrinol. Metab. 76, 112–116[Abstract]
  13. Milgrom, E. (1990) in Steroid Hormones (Baulieu, E-E., and Kelly, P. A., eds) pp. 398–402, Chapman and Hall, New York and London
  14. De la Torre, R., de la Torre, X., Alia, C., Segura, J., Baro, T., and Torres-Rodriguez, J. M. (2001) Anal. Biochem. 289, 116–123[CrossRef][Medline] [Order article via Infotrieve]
  15. Pedersen, L. C., Petrotchenko, E. V., and Negishi, M. (2000) FEBS Lett. 475, 61–64[CrossRef][Medline] [Order article via Infotrieve]
  16. Rehse, P., Zhou, M., and Lin, S. X. (2002) Biochem. J. 364, 165–171[Medline] [Order article via Infotrieve]
  17. Chang, H. J., Zhou, M., and Lin, S. X. (2001) J. Steroid Biochem. Mol. Biol. 77, 159–165[CrossRef][Medline] [Order article via Infotrieve]
  18. Cornish-Bowden, A. (1995) Analysis of Enzyme Kinetic Data, pp. 118–122, Oxford University Press, Oxford
  19. Gangloff, A., Garneau, A., Huang, Y. W., and Lin, S. X. (2001) Biochem. J. 356, 269–276[CrossRef][Medline] [Order article via Infotrieve]
  20. Zhou, M., Rehse, P., Chang, H. J., Luu-The, V., and Lin, S. X. (2001) Acta Crystallogr. D. Biol. Crystallogr. Sect. D 57, 1630–1633[CrossRef]
  21. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307–326[CrossRef]
  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., and Warren, G. L. (1998) Acta Crystallogr. D. Biol. Crystallogr. 54, 905–921[CrossRef][Medline] [Order article via Infotrieve]
  23. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. A Fund. Crystallogr. 47, 110–119[CrossRef]
  24. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283–291[CrossRef]
  25. Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N., and Bourne, P. E. (2000) Nucleic Acids Res. 28, 235–242[Abstract/Free Full Text]
  26. Otterness, D. M., Wieben, E. D., Wood, T. C., Waston, R. W. G., Madden, B. J., McCormick, D. J., and Weinshiboum, R. M. (1992) Mol. Pharmacol. 41, 865–872[Abstract]
  27. Sugiyama, Y., Stolz, A., Sugimoto, M., Kuhlenkamp, J., Yamada, T., and Kaplowitz, N. (1984) Biochem. J. 224, 947–953[Medline] [Order article via Infotrieve]
  28. Marcus, C. J., Sekura, R. D., and Jakoby, W. B. (1980) Anal. Biochem. 107, 296–304[CrossRef][Medline] [Order article via Infotrieve]
  29. Driscoll, W. J., Martin, B. M., Chen, H. C., and Strott, C. A. (1993) J. Biol. Chem. 268, 23496–23503[Abstract/Free Full Text]
  30. Park, B. C., Lee, Y. C., and Strott, C. A. (1999) J. Biol. Chem. 274, 21562–21568[Abstract/Free Full Text]
  31. Zhorov, B. S., and Lin, S. X. (2000) Proteins 38, 414–427[CrossRef][Medline] [Order article via Infotrieve]
  32. Zhang, H., Varmalova, O., Vargas, F. M., Falany, C. N., and Leyh, T. S. (1998) J. Biol. Chem. 273, 10888–10892[Abstract/Free Full Text]
  33. Luu-The, V., Bernier, F., and Dufort, I. (1996) J. Endocrinol. 150, S87-S97[Medline] [Order article via Infotrieve]
  34. Dickerman, Z., Grant, D. R., Faiman, C., and Winter, J. S. D. (1984) J. Clin. Endocrinol. Metab. 59, 1031–1036[Abstract/Free Full Text]
  35. Evans, S. V. (1993) J. Mol. Graphics 11, 134–138[CrossRef][Medline] [Order article via Infotrieve]
  36. Harris, M., and Jones, T. A. (2001) Acta Crystallogr. D Biol. Crystallogr. 57, 1201–1203[CrossRef][Medline] [Order article via Infotrieve]
  37. Wallace, A. C., Laskowski, R. A., and Thornton, J. M. (1995) Prot. Eng. 8, 127–134[Abstract/Free Full Text]

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