Testing the Sulfotransferase Molecular Pore Hypothesis

Human cytosolic sulfotransferases (SULTs) regulate the activities of hundreds of signaling metabolites via transfer of the sulfuryl moiety (-SO₃) from activated sulfate (3′-phosphoadenosine 5′-phosphosulfate) to the hydroxyls and primary amines of xeno- and endobiotics. How SULTs select substrates from the scores of competing ligands present in a cytosolic milieu is an important issue in the field. Selectivity appears to be sterically controlled by a molecular pore that opens and closes in response to nucleotide binding. This point of view is fostered by structures showing nucleotide-dependent pore closure and the fact that nucleotide binding induces an isomerization that restricts access to the acceptor-binding pocket. Molecular dynamics models underscore the importance of pore isomerization in selectivity and predict that specific molecular linkages stabilize the closed pore in response to nucleotide binding. To test the pore model, these linkages were disrupted in SULT2A1 via mutagenesis, and the effects on selectivity were determined. The mutations uncoupled nucleotide binding from selectivity and produced enzymes that no longer discriminated between large and small substrates. The mutations did not affect the affinity or turnover of small substrates but resulted in a 183-fold gain in catalytic efficiently toward large substrates. Models predict that an 11-residue “flap” covering the acceptor-binding pocket can open and admit large substrates when nucleotide is bound; a mutant structure demonstrated that this is so. In summary, the model was shown to be a robust, accurate predictor of SULT structure and selectivity whose general features will likely apply to other members of the SULT family.     

Crystal structures of SULT1A2 and SULT1A1∗3: Insights into the substrate inhibition and the role of Tyr149 in SULT1A2

The cytosolic sulfotransferases (SULTs) in vertebrates catalyze the sulfonation of endogenous thyroid/steroid hormones and catecholamine neurotransmitters, as well as a variety of xenobiotics, using 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as the sulfonate donor. In this study, we determined the structures of SULT1A2 and an allozyme of SULT1A1, SULT1A1∗3, bound with 3′-phosphoadenosine 5′-phosphate (PAP), at 2.4 and 2.3 Å resolution, respectively. The conformational differences between the two structures revealed a plastic substrate-binding pocket with two channels and a switch-like substrate selectivity residue Phe247, providing clearly a structural basis for the substrate inhibition. In SULT1A2, Tyr149 extends approximately 2.1 Å further to the inside of the substrate-binding pocket, compared with the corresponding His149 residue in SULT1A1∗3. Site-directed mutagenesis study showed that, compared with the wild-type SULT1A2, mutant Tyr149Phe SULT1A2 exhibited a 40 times higher K_m and two times lower V_max with p-nitrophenol as substrate. These latter data imply a significant role of Tyr149 in the catalytic mechanism of SULT1A2.     

Isotope exchange at equilibrium indicates a steady state ordered kinetic mechanism for human sulfotransferase

Cytosolic sulfotransferase (SULT)-catalyzed sulfation regulates biosignaling molecular biological activities and detoxifies hydroxyl-containing xenobiotics. The universal sulfuryl group donor for SULTcatalyzed sulfation is adenosine 3'-phosphate 5'-phosphosulfate (PAPS). The reaction products are a sulfated product and adenosine 3',5'-diphosphate (PAP). Although the kinetics has been reported since the 1980s,SULT-catalyzed reaction mechanisms remain unclear. Human SULT1A1 catalyzes the sulfation of xenobiotic phenols and has very broad substrate specificity. It has been recognized as one of the most important phase II drug-metabolizing enzymes. Understanding the kinetic mechanism of this isoform is important in understanding drug metabolism and xenobiotic detoxification. In this report, we investigated the SULT1A1-catalyzed phenol sulfation mechanism. The SULT1A1-catalyzed reaction was brought to equilibrium by varying substrate (1-naphthol) and PAPS initial concentrations. Equilibrium constants were determined. Two isotopic exchanges at equilibrium ([14C]1-naphthol <=>[14C]1-naphthyl sulfate and[35S]PAPS<=>[35S]1-naphthyl sulfate) were conducted. First-order kinetics, observed for all the is otopic exchange reactions studied over the entire time scale that was monitored, indicates that the system was truly at equilibrium prior to addition of an isotopic pulse. Complete suppression of the 35S isotopic exchange rate was observed with an increase in the levels of 1-naphthol and 1-naphthyl sulfate in a constant ratio,while no suppression of the 14C exchange rate was observed with an increase in the levels of PAPS and PAP in a constant ratio. Data are consistent with a steady state ordered kinetic mechanism with PAPS and PAP binding to the free enzyme.     

Interactions of the human cytosolic sulfotransferases and steroid sulfatase in the metabolism of tibolone and raloxifene

Sulfation is important in the metabolism and inactivation of steroidal compounds and hormone replacement therapeutic (HRT) agents in human tissues. Although generally inactive, many steroid sulfates are hydrolyzed to their active forms by sulfatase activity. Therefore, the specific sulfotransferase (SULT) isoforms and the levels of steroid sulfatase (STS) activity in tissues are important in regulating the activity of steroidal and HRT compounds. Tibolone (Tib) is metabolized to three active metabolites and all four compounds are readily sulfated. Tib and the Δ4-isomer are sulfated at the 17β-OH group by SULT2A1 and the 17-sulfates are resistant to hydrolysis by human placental STS. 3-OH and 3β-OH Tib can form both 3- and 17-monosulfates as well as disulfates. Only the 3β-sulfates are susceptible to STS hydrolysis. Raloxifene monosulfation was catalyzed by at least seven SULT isoforms and SULT1E1 also synthesizes raloxifene disulfate. SULT1E1 forms both monosulfates in a ratio of approximately 8:1 with the more abundant monosulfate migrating on HPLC identical to the SULT2A1 synthesized monosulfate. The raloxifene monosulfate formed by both SULT isoforms is sensitive to STS hydrolysis whereas the low abundance monosulfate formed by SULT1E1 is resistant. The benzothiophene sulfates of raloxifene and arzoxifene were hydrolyzed by STS whereas the raloxifene 4′-phenolic sulfate was resistant. These results indicate that tissue specific expression of SULT isoforms and STS could be important in the inactivation and regeneration of the active forms of HRT agents.     

The molecular basis for the broad substrate specificity of human sulfotransferase 1A1

Cytosolic sulfotransferases (SULTs) are mammalian enzymes that detoxify a wide variety of chemicals through the addition of a sulfate group. Despite extensive research, the molecular basis for the broad specificity of SULTs is still not understood. Here, structural, protein engineering and kinetic approaches were employed to obtain deep understanding of the molecular basis for the broad specificity, catalytic activity and substrate inhibition of SULT1A1. We have determined five new structures of SULT1A1 in complex with different acceptors, and utilized a directed evolution approach to generate SULT1A1 mutants with enhanced thermostability and increased catalytic activity. We found that active site plasticity enables binding of different acceptors and identified dramatic structural changes in the SULT1A1 active site leading to the binding of a second acceptor molecule in a conserved yet non-productive manner. Our combined approach highlights the dominant role of SULT1A1 structural flexibility in controlling the specificity and activity of this enzyme.     

Substrate inhibition in human hydroxysteroid sulfotransferase SULT2A1: studies on the formation of catalytically non-productive enzyme complexes

The cytosolic sulfotransferase hSULT2A1 is the major hydroxysteroid (alcohol) sulfotransferase in human liver, and it catalyzes the 3′-phosphoadenosine-5′-phosphosulfate (PAPS)-dependent sulfation of various endogenous hydroxysteroids as well as many xenobiotics that contain alcohol and phenol functional groups. The hSULT2A1 often displays substrate inhibition, and we have hypothesized that a key element in this response to increasing substrate concentration is the formation of non-productive ternary dead-end enzyme complexes involving the nucleotide product, adenosine 3′,5′-diphosphate (PAP). One of these substrates for hSULT2A1 is dehydroepiandrosterone (DHEA), a major circulating steroid hormone in humans that serves as precursor to both androgens and estrogens. We have utilized DHEA in both initial velocity studies and equilibrium binding experiments in order to evaluate the potential role of ternary complexes in substrate inhibition of the enzyme. Our results indicate that hSULT2A1 forms non-productive ternary complexes that involve either DHEA or dehydroepiandrosterone sulfate, and the formation of these ternary complexes displays negative cooperativity in the binding of DHEA.     

Arginine residues in the active site of human phenol sulfotransferase (SULT1A1)

Cytosolic sulfotransferases (STs) catalyze the sulfation of hydroxyl containing compounds. Human phenol sulfotransferase (SULT1A1) is the major human ST that catalyzes the sulfation of simple phenols. Because of its broad substrate specificity and lack of endogenous substrates, the biological function of SULT1A1 is believed to be an important detoxification enzyme. In this report, amino acid modification, computer structure modeling, and site-directed mutagenesis were used for studies of Arg residues in the active site of SULT1A1. The Arg-specific modification reagent, 2,3-butanedione, inactivated SULT1A1 in an efficient, time- and concentration-dependent manner, suggesting Arg residues play an important role in the catalytic activity of SULT1A1. According to the computer model, Arg78, Arg130, and Arg257 may be important for SULT1A1 catalytic activity. Site-directed mutagenesis results demonstrated that the positive charge on Arg78 is not critical for SULT1A1 because R78A is still active. In contrast, a negative charge at this position, R78E, completely inactivated SULT1A1. Arg78 is in close proximity to the site of sulfuryl group transfer. Arg257 is located very close to the 3'-phosphate in adenosine 3'-phosphate 5'-phosphosulfate (PAPS). Site-directed mutagenesis demonstrated that Arg257 is critical for SULT1A1: both R257A and R257E are inactive. Although Arg130 is also located very close to the 3'-phosphate of PAPS, R130A and R130E are still active, suggesting that Arg130 is not a critical residue for the catalytic activity of SULT1A1. Computer modeling suggests that the ionic interaction between the positive charge on Arg257, and the negative charge on 3'-phosphate is the primary force stabilizing the specific binding of PAPS.     

Sulfation of iodothyronines by recombinant human liver steroid sulfotransferases.

Sulfation is an important pathway in the metabolism of thyroid hormones. Sulfated iodothyronines are elevated in nonthyroidal illnesses and in the normal human fetal circulation. We assayed and characterized COS-1 cell expressed recombinant human liver dehydroepiandrosterone sulfotransferase (DHEA ST or SULT2A1) and estrogen sulfotransferase (EST or SULT1E1) activities for the first time with triiodothyronine (T(3)) as the substrate. Several biochemical properties that included apparent K(m) values, thermal stabilities, and responses to the inhibitors 2, 6-dichloro-4-nitrophenol and NaCl were tested. SULT2A1, a member of the hydroxysteroid sulfotransferase family, used 3,3'-T(2) more readily than T(3) and 3,5-T(2) as substrates, but had the lowest apparent K(m) value for T(3) of any reported human SULT. SULT1E1, a member of the phenol sulfotransferase family, used 3,3'-T(2) and rT(3) more readily than T(3), and also displayed the greatest specificity for T(4) among human SULTs. SULT2A1 may contribute more to iodothyronine sulfation than previously suspected. Potential roles of both steroid sulfotransferases in the enhanced sulfation of nonthyroidal illnesses and fetal development invite further investigation.     

Assay of sulfotransferases.

Two methods are described for the assay of sulfotransferases which are active with sulfate acceptors bearing the hydroxyl functional group. Assays were developed for enzymes which transfer sulfate from 3′-phosphoadenosine–5′-phosphosulfate (PAPS) to sterols, phenols, and simple alcohols thereby forming the corresponding sulfate esters. With a filter binding assay, useful with crude and purified enzyme preparations, a radioactive sterol substrate is used and subsequently separated from labeled product, allowing the determination of between 50 and 400 pmol of product. In a second method, [³⁵S]PAPS is used and the labeled product is separated from PAPS and inorganic sulfate by a thin-layer technique in which product migrates close to the solvent front; the assay is useful with a broad array of substrates and is more sensitive than the filter binding assay.     

Substrate-induced conformational changes in glycosyltransferases

Oligosaccharide chains of glycoproteins, glycolipids and glycosaminoglycans are synthesized by glycosyltransferases by the transfer of specific glycosyl moieties from activated sugar-nucleotide donors to specific acceptors. Structural studies on several of these enzymes have shown that one or two flexible loops at the substrate-binding site of the enzymes undergo a marked conformational change from an open to a closed conformation on binding the donor substrate. This conformational change, in which the loop acts as a lid covering the bound donor substrate, creates an acceptor-binding site. After the glycosyl unit is transferred from the donor to the acceptor, the saccharide product is ejected and the loop reverts to its native conformation, thereby releasing the remaining nucleotide moiety. The specificity of the sugar donor is determined by a few residues in the sugar-nucleotide-binding pocket of the enzyme that are conserved among the family members from different species.     

Reaction product affinity regulates activation of human sulfotransferase 1A1 PAP sulfation

Cytosolic sulfotransferase (SULT)-catalyzed sulfation regulates the activity of bio-signaling molecules and aids in metabolizing hydroxyl-containing xenobiotics. The sulfuryl donor for the SULT reaction is adenosine 3′-phosphate 5′-phosphosulfate (PAPS), while products are adenosine 3′,5′-diphosphate (PAP) and a sulfated alcohol. Human phenol sulfotransferase (SULT1A1) is one of the major detoxifying enzymes for phenolic xenobiotics. The mechanism of SULT1A1-catalyzed sulfation of PAP by pNPS was investigated. PAP was sulfated by para-nitrophenyl sulfate (pNPS) in a concentration-dependent manner. 2-Naphthol inhibited sulfation of PAP, competing with pNPS, while phenol activated the sulfation reaction. At saturating PAP, a ping pong kinetic mechanism is observed with pNPS and phenol as substrates, consistent with phenol intercepting the E–PAPS complex prior to dissociation of PAPS. At high concentrations, phenol competes with pNPS, consistent with formation of the E–PAP–phenol dead-end complex. Data are consistent with the previously reported mechanism for sulfation of 2-naphthol by PAPS, and its activation by pNPS [14]. Overall, data are consistent with release of PAP from E–PAP and PAPS from E–PAPS contributing to rate-limitation in both reaction directions.     

Spectrofluorimetric analysis of 7-hydroxycoumarin binding to bovine phenol sulfotransferase

Phenol sulfotransferases (SULT1s, EC 2.8.2.1) catalyze sulfuryl group transfer from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the hydroxyl oxygen of aromatic acceptor substrates. Previous work with the bovine SULT1A1 has utilized the highly fluorescent substrate 7-hydroxycoumarin (7-HC, umbelliferone) as an acceptor substrate [Biochem. Biophys. Res. Commun. 261 (1999) 815]. Here we report that adenosine-3',5'-bisphosphate (PAP)-dependent binding of 7-HC to bSULT1A1 can be observed due to the appearance of a 400-420-nm shoulder in the emission spectrum, using an excitation wavelength of 280 nm. This emission was observed by placing 7-HC in ethanol, which is consistent with bSULT1A1 phenol binding site hydrophobicity. Titrations with 7-HC indicate a K(d) for 7-HC of 0.58 microM and substoichiometric binding to the homodimeric enzyme. The bSULT1A1:PAP:7-HC complex could be disrupted with pentachlorophenol (PCP), titrations with which indicated 0.5 equivalents per enzyme subunit. Titrations of enzyme plus 7-HC with PAP also indicated 0.5 equivalents per enzyme subunit. These results suggest a model of homodimeric bSULT1A1 in which subunit interactions favor half-site reactivity in the formation of a dead end complex.     

Crystal structure-based studies of cytosolic sulfotransferase

Sulfation is a widely observed biological reaction conserved from bacterium to human that plays a key role in various biological processes such as growth, development, and defense against adversities. Deficiencies due to the lack of the ubiquitous sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) are lethal in humans. A large group of enzymes called sulfotransferases catalyze the transfer reaction of sulfuryl group of PAPS to the acceptor group of numerous biochemical and xenochemical substrates. Four X-ray crystal structures of sulfotransferases have now been determined: cytosolic estrogen, hydroxysteroid, aryl sulfotransferases, and a sulfotransferase domain of the Golgi-membrane heparan sulfate N-deacetylase/N-sulfotransferase 1. These have revealed the conserved core structure of the PAPS binding site, a common reaction mechanism, and some information concerning the substrate specificity. These crystal structures introduce a new era of the study of the sulfotransferases.     

Correlation between PAP-dependent steroid binding activity and substrate specificity of mouse and human estrogen sulfotransferases

Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the sulfoconjugation and inactivation of estrogens using 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as an activated sulfate donor. A finding of undetermined significance in the study of EST has been that the guinea pig EST is able to bind pregnenolone and estradiol with high affinity in the presence of PAP, the reaction by-product of the sulfate donor PAPS. This finding has raised the possibility that EST may have other physiological functions independent of its enzymatic activity as a sulfotransferase. To determine if the PAP-dependent steroid binding activity is a common property shared by other estrogen sulfotransferases, we have expressed the mouse and human EST in bacteria and used the purified protein to address this question. We found that, in the presence of PAP, both recombinant mouse and human EST were able to bind estradiol with high affinity but only the human EST was able to bind pregnenolone. In addition, we show that human but not the mouse EST was also able to bind dehydroepiandrosterone, a property that was not described for the guinea pig EST. Furthermore, we demonstrate that the promiscuity of human EST in steroid binding is mirrored by a correspondingly low substrate specificity in its enzymatic activity as a sulfotransferase. Reversely, the lack of stable binding of pregnenolone and dehydroepiandrosterone by the mouse EST is paralleled by a lack of sulfotransferase activity of this enzyme toward these two steroids. Mutagenesis of mouse EST within a domain critical for PAPS binding abolished both its sulfotransferase and PAP-dependent estrogen binding activity. These data suggest that stable binding of steroids such as pregnenolone or estrogen is not an independent property of estrogen sulfotransferases but rather is related to their catalytic activity.     

Sulfation of environmental estrogen-like chemicals by human cytosolic sulfotransferases

To investigate whether sulfation, a major Phase II detoxification pathway in vivo, can be employed as a means for the inactivation/disposal of environmental estrogens, recombinant human cytosolic sulfotransferases were prepared and tested for enzymatic activities with bisphenol A, diethylstilbestrol, 4-octylphenol, p-nonylphenol, and 17alpha-ethynylestradiol as substrates. Of the seven recombinant enzymes examined, only SULT1C sulfotransferase #1 showed no activities toward the environmental estrogens tested. Among the other six sulfotransferases, the simple phenol (P)-form phenol sulfotransferase and estrogen sulfotransferase appeared to be considerably more active toward environmental estrogens than the other four sulfotransferases. Metabolic labeling experiments revealed the sulfation of environmental estrogens and the release of their sulfated derivatives by HepG2 human hepatoma cells. Moreover, sulfated environmental estrogens appeared to be incapable of penetrating through the HepG2 cell membrane.     

In vitro and in vivo effects of a cyclooxygenase-2 inhibitor nimesulide analog JCC76 in aromatase inhibitors-insensitive breast cancer cells

Glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGT) and sulfation, catalyzed by sulfotransferases (SULT), are pathways through which sex steroids are metabolized to less active compounds. These enzymes are highly polymorphic and genetic variants frequently result in higher or lower activity. The phenotypic effects of these polymorphisms on circulating sex steroids in premenopausal women have not yet been investigated. One hundred and seventy women aged 40-45 years had a blood sample drawn during the follicular phase of the menstrual cycle for sex steroid measures and to obtain genomic DNA. Urine was collected for 2-hydroxy (OH) estrone (E(1)) and 16α-OH E(1) measures. Generalized linear regression models were used to assess associations between sex steroids and polymorphisms in the UGT1A and UGT2B families, SULT1A1, and SULT1E1. Women with the UGT1A1(TA7/TA7) genotype had 25% lower mean estradiol (E(2)) concentrations compared to the wildtype (TA6/TA6) (p=0.02). Similar associations were observed between SULT1A1(R213/H213) and E(1) (13% lower mean E(1) concentration vs. wildtype; p-value=0.02) and UGT2B4(E458/E458) and dehydroepiandrosterone (DHEA) (20% lower mean DHEA vs. wildtype; p-value=0.03). The SULT1E1(A/C) and the UGT1A1(TA7)-UGT1A3(R11) haplotypes were associated with reduced estrogen concentrations. Further study of UGT and SULT polymorphisms and circulating sex steroid measures in larger populations of premenopausal women is warranted.     

Identification of novel hydroxysteroid-sulfating cytosolic SULTs, SULT2 ST2 and SULT2 ST3, from zebrafish: cloning, expression, characterization, and developmental expression

By searching the expressed sequence tag database, two zebrafish cDNAs encoding putative cytosolic sulfotransferases (SULTs) were identified. Sequence analysis indicated that these two zebrafish SULTs belong to the cytosolic SULT2 gene family. The recombinant form of these two novel zebrafish SULTs, designated SULT2 ST2 and SULT2 ST3, were expressed using the pGEX-2TK glutathione S-transferase (GST) gene fusion system and purified from transformed BL21 (DE3) Escherichia coli cells. Purified GST-fusion protein form of SULT2 ST2 and SULT2 ST3 exhibited strong sulfating activities toward dehydroepiandrosterone (DHEA) and corticosterone, respectively, among various endogenous compounds tested as substrates. Both enzymes displayed pH optima at approximately 6.5. Kinetic constants of the two enzymes, as well as the GST-fusion protein form of the previously identified SULT2 ST1, with DHEA and corticosterone as substrates were determined. Developmental stage-dependent expression experiments revealed distinct patterns of expression of SULT2 ST2 and SULT2 ST3, as well as the previously identified SULT2 ST1, during embryonic development and throughout the larval stage onto maturity.