A new type of sulfation reaction: C-sulfonation for α,ß-unsaturated carbonyl groups by a novel sulfotransferase SULT7A1.

Cytosolic sulfotransferases (SULTs) are cytosolic enzymes that catalyze the transfer of sulfonate group to key endogenous compounds, altering the physiological functions of their substrates. SULT enzymes catalyze the O-sulfonation of hydroxy groups or N-sulfonation of amino groups of substrate compounds. In this study, we report the discovery of C-sulfonation of α,ß-unsaturated carbonyl groups mediated by a new SULT enzyme, SULT7A1, and human SULT1C4. Enzymatic assays revealed that SULT7A1 is capable of transferring the sulfonate group from 3'-phosphoadenosine 5'-phosphosulfate to the α-carbon of α,ß-unsaturated carbonyl-containing compounds, including cyclopentenone prostaglandins as representative endogenous substrates. Structural analyses of SULT7A1 suggest that the C-sulfonation reaction is catalyzed by a novel mechanism mediated by His and Cys residues in the active site. Ligand-activity assays demonstrated that sulfonated 15-deoxy prostaglandin J2 exhibits antagonist activity against the prostaglandin receptor EP2 and the prostacyclin receptor IP. Modification of α,ß-unsaturated carbonyl groups via the new prostaglandin-sulfonating enzyme, SULT7A1, may regulate the physiological function of prostaglandins in the gut. Discovery of C-sulfonation of α,ß-unsaturated carbonyl groups will broaden the spectrum of potential substrates and physiological functions of SULTs.

Evolution and multiple functions of sulfonation and cytosolic sulfotransferases across species.

Organisms have conversion systems for sulfate ion to take advantage of the chemical features. The use of biologically converted sulfonucleotides varies in an evolutionary manner, with the universal use being that of sulfonate donors. Sulfotransferases have the ability to transfer the sulfonate group of 3'-phosphoadenosine 5'-phosphosulfate to a variety of molecules. Cytosolic sulfotransferases (SULTs) play a role in the metabolism of low-molecular-weight compounds in response to the host organism's living environment. This review will address the diverse functions of the SULT in evolution, including recent findings. In addition to the diversity of vertebrate sulfotransferases, the molecular aspects and recent studies on bacterial and plant sulfotransferases are also addressed.

Effects of sturgeon fillet intake on top-ranked Japanese female long-distance runners.

AIMS: The aim of this study is to investigate whether consumption of sturgeon fillets reduces the oxidative stress marker urinary 8-hydroxy-2'-deoxyguanosine (8OHdG) in top-ranked Japanese female long-distance runners. METHODS: In a before-and-after study, nine professional long-distance female athletes ate 100 g/day of sturgeon fillets for 2 weeks. Urinalysis (8OHdG, an oxidative stress marker, and creatinine), blood tests (fatty acids and 25-hydroxyvitamin D [25OHD]), exercise intensity, subjective fatigue, muscle elasticity, muscle mass, body fat mass, and nutritional intake using image-based dietary assessment (IBDA) were compared before, immediately after, and 1 month after the intervention. RESULTS: Consumption of sturgeon fillets suppressed 8OHdG (p < 0.05) in the increased exercise intensity female athletes. Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and 25OHD levels in blood increased from before to immediately after and 1 month after the intervention (p < 0.05). IBDA showed that intake of n-3 fatty acid increased after and one month after the intervention, whereas DHA, imidazole dipeptide and vitamin D intake increased after the intervention (p < 0.05) and then decreased after 1 month (p < 0.05). There were no significant changes in subjective fatigue, muscle elasticity, muscle mass, and body fat. CONCLUSIONS: The results suggest that eating sturgeon fillets during intense training may increase blood levels of EPA, DHA, and 25OHD, which may suppress urinary oxidative stress (8OHdG) in top-ranked Japanese long-distance runners.

Investigation of radical scavenging effects of acetaminophen, p-aminophenol and their O-sulfated conjugates.

Acetaminophen (APAP) and p-aminophenol (p-AP) are the analogous simple phenolic compounds that undergo sulfate conjugation (sulfation) by cytosolic sulfotransferases. Sulfation is generally thought to lead to the inactivation and disposal of endogenous as well as xenobiotic compounds. This study aimed to investigate the antioxidative effects of O-sulfated form of APAP and p-AP, i.e., APAPS and p-APS, in comparison with their unsulfated counterparts. Using a 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay, the antioxidant capacity of APAPS was shown to be approximately 126-times lower than that of APAP. In contrast, p-APS displayed comparable activity as unsulfated p-AP. Similar trends concerning the suppressive effects of these chemicals on cellular O2- radical generation were found using an activated granulocytic neutrophil cell model. Collectively, these results indicated that, depending on the presence of an additional "active site", sulfation may not always decrease the antioxidant activities of phenolic compounds.

Sulfation of 12-hydroxy-nevirapine by human SULTs and the effects of genetic polymorphisms of SULT1A1 and SULT2A1.

Nevirapine (NVP) is an effective drug for the treatment of HIV infections, but its use is limited by a high incidence of severe skin rash and liver injury. 12-Hydroxynevirapine (12-OH-NVP) is the major metabolite of nevirapine. There is strong evidence that the sulfate of 12-OH-NVP is responsible for the skin rash. While several cytosolic sulfotransferases (SULTs) have been shown to be capable of sulfating 12-OH-NVP, the exact mechanism of sulfation in vivo is unclear. The current study aimed to clarify human SULT(s) and human organs that are capable of sulfating 12-OH-NVP and investigate the metabolic sulfation of 12-OH-NVP using cultured HepG2 human hepatoma cells. Enzymatic assays revealed that of the thirteen human SULTs, SULT1A1 and SULT2A1 displayed strong 12-OH-NVP-sulfating activity. 1-Phenyl-1-hexanol (PHHX), which applied topically prevents the skin rash in rats, inhibited 12-OH-NVP sulfation by SULT1A1 and SULT2A1, implying the involvement of these two enzymes in the sulfation of 12-OH-NVP in vivo. Among five human organ cytosols analyzed, liver cytosol displayed the strongest 12-OH-NVP-sulfating activity, while a low but significant activity was detected with skin cytosol. Cultured HepG2 cells were shown to be capable of sulfating 12-OH-NVP. The effects of genetic polymorphisms of SULT1A1 and SULT2A1 genes on the sulfation of 12-OH-NVP by SULT1A1 and SULT2A1 allozymes were investigated. Two SULT1A1 allozymes, Arg37Asp and Met223Val, showed no detectable 12-OH-NVP-sulfating activity, while a SULT2A1 allozyme, Met57Thr, displayed significantly higher 12-OH-NVP-sulfating activity compared with the wild-type enzyme. Collectively, these results contribute to a better understanding of the involvement of sulfation in NVP-induced skin rash and provide clues to the possible role of SULT genetic polymorphisms in the risk of this adverse reaction.

Novel silkworm (Bombyx mori) sulfotransferase swSULT ST3 is involved in metabolism of polyphenols from mulberry leaves.

Polyphenols in plants are important for defense responses against microorganisms, insect herbivory, and control of feeding. Owing to their antioxidant, anti-cancer, and anti-inflammatory activities, their importance in human nutrition has been acknowledged. However, metabolism of polyphenols derived from mulberry leaves in silkworms (Bombyx mori) remains unclear. Sulfotransferases (SULT) are involved in the metabolism of xenobiotics and endogenous compounds. The purpose of this study is to investigate the metabolic mechanism of polyphenols mediated by B. mori SULT. Here, we identified a novel SULT in silkworms (herein, swSULT ST3). Recombinant swSULT ST3 overexpressed in Escherichia coli effectively sulfated polyphenols present in mulberry leaves. swSULT ST3 showed high specific activity toward genistein among the polyphenols. Genistein-7-sulfate was produced by the activity of swSULT ST3. Higher expression of swSULT ST3 mRNA was observed in the midgut and fat body than in the hemocytes, testis, ovary, and silk gland. Polyphenols inhibited the aldo-keto reductase detoxification of reactive aldehydes from mulberry leaves, and the most noticeable inhibition was observed with genistein. Our results suggest that swSULT ST3 plays a role in the detoxification of polyphenols, including genistein, and contributes to the effects of aldo-keto reductase in the midgut of silkworms. This study provides new insight into the functions of SULTs and the molecular mechanism responsible for host plant selection in lepidopteran insects.

Molecular cloning and characterization of common marmoset SULT1C subfamily members that catalyze the sulfation of thyroid hormones.

Cytosolic sulfotransferase SULT1C subfamily is one of the most flexible gene subfamilies during mammalian evolution. The physiological functions of SULT1C enzymes still remain to be fully understood. In this study, common marmoset (Callithrix jacchus), a promising primate animal model, was used to investigate the functional relevance of the SULT1C subfamily. Gene database search revealed 3 intact SULT1C genes and a pseudogene in its genome. These 4 genes were named SULT1C1, SULT1C2, SULT1C3P, and SULT1C5, according to the sequence homology and gene location. Since SULT1C5 is the orthologous gene for human SULT1C2P, we propose, here, to revisit the designation of human SULT1C2P to SULT1C5P. Purified recombinant SULT1C enzymes showed sulfating activities toward a variety of xenobiotic compounds and thyroid hormones. Kinetic analysis revealed high catalytic activities of SULT1C1 and SULT1C5 for 3,3'-T2. It appears therefore that SULT1C isoforms may play a role in the thyroid hormone metabolism in common marmoset.

The crystal structure of mouse SULT2A8 reveals the mechanism of 7α-hydroxyl, bile acid sulfation.

Bile acids play essential roles in facilitating the intestinal absorption of lipophilic nutrients as well as regulation of glucose, lipid, and energy homeostasis via activation of some receptors. Bile acids are cytotoxic, and consequently their concentrations are tightly controlled. A critical pathway for bile acid elimination and detoxification is sulfation. The pattern of bile acid sulfation differs by species. Sulfation preferentially occurs at the 3α-OH of bile acids in humans, but at the 7α-OH in mice. A recent study identified mouse cytosolic sulfotransferase 2A8 (mSULT2A8) as the major hepatic 7α-hydroxyl bile acid-sulfating enzyme. To elucidate the 7α-OH specific sulfation mechanism of mSULT2A8, instead of 3α-OH specific sulfation in humans, we determined a crystal structure of mSULT2A8 in complex with cholic acid, a major bile acid, and 3'-phosphoadenosine-5'-phosphate, the sulfate donor product. Our study shows that bile acid-binding mode of mSULT2A8 and how the enzyme holds the 7α-OH group of bile acids at the catalytic center, revealing that the mechanism underlying 7α-OH specific sulfation. The structure shows the substrate binds to mSULT2A8 in an orientation perpendicular to that of human 3α-hydroxyl bile acid-sulfotransferase (hSULT2A1). The structure of the complex provides new insight into species different bile acid metabolism.

Proteolytic Maturation of the Outer Membrane c-Type Cytochrome OmcZ by a Subtilisin-Like Serine Protease Is Essential for Optimal Current Production by Geobacter sulfurreducens.

An outer membrane c-type cytochrome (OmcZ) in Geobacter sulfurreducens is essential for optimal current production in microbial fuel cells. OmcZ exists in two forms, small and large, designated OmcZS and OmcZL, respectively. However, it is still not known how these two structures are formed. A mutant with a disruption of the GSU2075 gene encoding a subtilisin-like serine protease (designated ozpA for the OmcZprotease), which is located downstream of omcZ, produced low currents at a level similar to that of the omcZ-deficient mutant strain. Biochemical analyses revealed that the ozpA mutant accumulated OmcZL and did not produce OmcZS, which is thought to be a mature form that is essential for the extracellular electron transfer to the electrode. A heterologous expression system cell lysate from an Escherichia coli strain producing OzpA cleaved OmcZL and generated OmcZS as the proteolytic product. Among the culture supernatant, loosely bound outer surface, and intracellular protein fractions from wild-type G. sulfurreducens, only the culture supernatant protein fraction showed OmcZL cleavage activity, indicating that the mature form of OmcZ, OmcZS, can be produced outside the cells. These results indicate that OzpA is an essential protease for current production via the maturation of OmcZ, and OmcZS is the key to the extracellular electron transfer to electrodes. This proteolytic maturation of OmcZ is a unique regulation among known c-type cytochromes in G. sulfurreducens. IMPORTANCE Microbial fuel cells are a promising technology for energy generation from various waste types. However, the molecular mechanisms of microbial extracellular electron transfer to the electrode need to be elucidated. G. sulfurreducens is a common key player in electricity generation in mixed-culture microbial fuel cell systems and a model microorganism for the study of extracellular electron transfer. Outer membrane c-type cytochrome OmcZ is essential for an optimal current production by G. sulfurreducens. OmcZ proteolytic cleavage occurs during maturation, but the underlying mechanism is unknown. This study identifies a subtilisin-like protease, OzpA, which plays a role in cleaving OmcZ and generating the mature form of OmcZ (OmcZS). OzpA is essential for current production and, thus, the proteolytic maturation of OmcZ. This is a novel regulation of the c-type cytochrome for G. sulfurreducens extracellular electron transfer. This study also provides new insights into the design strategy and development of microbial extracellular electron transfer for an efficient energy conversion from chemical energy to electricity.

Varietal differences in flavonoid and antioxidant activity in Japanese soybean accessions.

Soybean seeds contain many antioxidants, including flavonoids and other phytochemicals. Isoflavone is a phytoestrogen that mimics estrogenic effects on target tissues and also exerts antioxidant activity by sequestering free radicals. Despite many cultivars developed to date, varietal differences in flavonoid content and antioxidant activity in Japanese soybean accessions remain less well characterized. Here, we evaluated the seed content of isoflavones, total flavonoids, and total phenolics in 26 soybean accessions. Next, the antioxidant activity of each accession was examined using antioxidant responsive element (ARE) linked to a luciferase reporter in human HepG2 stable cells. The relative ARE luciferase activity rate of all soybean accessions varied up to 4-fold which ranged from 1.00 to 4.02; and 22 accessions exhibited significant antioxidant activities. Correlation analysis indicated that the level of total isoflavone moderately correlated with antioxidant activity.

Effects of indole and indoxyl on the intracellular oxidation level and phagocytic activity of differentiated HL-60 human macrophage cells.

Indoxyl, a derivative of indole originating from tryptophan, may undergo phase-II sulfate-conjugation pathway, thereby forming indoxyl sulfate (IS) in vivo. We previously reported that IS, a well-known uremic toxin, can increase the intracellular oxidation level and decrease the phagocytic activity in a differentiated HL-60 human macrophage cell model. Using the same cell model, the current study aimed to investigate whether indole and indoxyl (the metabolic precursors of indoxyl and IS, respectively) may cause macrophage immune dysfunction. Results obtained indicated that intracellular oxidation level and cytotoxicity markedly increased upon treatment with indole and indoxyl, in comparison with IS. Incubation of the cells with indole and indoxyl also resulted in attenuated phagocytic activity. Human serum albumin (HSA)-binding assay confirmed that tryptophan and IS, but not indole and indoxyl, could selectively bind to the site II in HSA. Collectively, the results indicated that indole and indoxyl may strongly down-regulate the phagocytic immune function of macrophages, whereas IS, formed upon sulfate conjugation of indoxyl, may exhibit enhanced HSA-binding capability, thereby reducing the adverse effects of indoxyl.

Investigation of the effects of indoxyl sulfate, a uremic toxin, on the intracellular oxidation level and phagocytic activity using an HL-60-differentiated human macrophage cell model.

Indoxyl sulfate (IS), a uremic toxin, is a sulfate-conjugated metabolite originated from tryptophan. Accumulating uremic toxins may worsen renal diseases and further complicate related disorders including impaired immune functions under oxidative stress conditions. However, it has remained unclear whether or not IS can directly cause the cellular immune dysfunction. We investigated the effects of IS on the intracellular oxidation level and phagocytic activity in a HL-60-differantiated human macrophage cell model. Incubation of the cells in the presence of IS resulted in increasing intracellular oxidation level and decreasing phagocytic activity. In addition to inhibitors for NADH oxidase (NOX), organic anion transporting polypeptide2B1 (OATP2B1), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K), a representative antioxidant Trolox, was also shown to significantly relieve the IS-induced oxidation and restore weakened phagocytosis. Collectively, IS may directly down-regulate the phagocytic immune function of macrophages through the oxidation mechanisms including OATP2B1, PKC, PI3K, and NOX pathways. Abbreviations: CKD: Chronic kidney disease; IS: Indoxyl sulfate; ROS: Reactive oxygen species; NOX: NADH oxidase; OATP2B1: Organic anion transporting polypeptide2B1; PKC: Protein kinase C; PI3K: Phosphoinositide 3-kinase; 2-APT: 2-acetylphenothiazine.

Effect of SULT2B1 genetic polymorphisms on the sulfation of dehydroepiandrosterone and pregnenolone by SULT2B1b allozymes.

Pregnenolone and dehydroepiandrosterone (DHEA) are hydroxysteroids that serve as biosynthetic precursors for steroid hormones in human body. SULT2B1b has been reported to be critically involved in the sulfation of pregnenolone and DHEA, particularly in the sex steroid-responsive tissues. The current study was designed to investigate the impact of the genetic polymorphisms of SULT2B1 on the sulfation of DHEA and pregnenolone by SULT2B1b allozymes. Ten SULT2B1b allozymes previously prepared were shown to exhibit differential sulfating activities toward DHEA and pregnenolone in comparison to the wild-type enzyme. Kinetic studies revealed further significant changes in their substrate-binding affinity and catalytic activity toward DHEA and pregnenolone. Taken together, these results indicated clearly a profound effect of SULT2B1 genetic polymorphisms on the sulfating activity of SULT2B1b allozymes toward DHEA and pregnenolone, which may have implications in inter-individual variations in the homeostasis of these two important steroid precursors.

Identification of zebrafish steroid sulfatase and comparative analysis of the enzymatic properties with human steroid sulfatase.

Steroid sulfatase (STS) plays an important role in the regulation of steroid hormones. Metabolism of steroid hormones in zebrafish has been investigated, but the action of steroid sulfatase remains unknown. In this study, a zebrafish sts was cloned, expressed, purified, and characterized in comparison with the orthologous human enzyme. Enzymatic assays demonstrated that similar to human STS, zebrafish Sts was most active in catalyzing the hydrolysis of estrone-sulfate and estradiol-sulfate, among five steroid sulfates tested as substrates. Kinetic analyses revealed that the Km values of zebrafish Sts and human STS differed with respective substrates, but the catalytic efficiency as reflected by the Vmax/Km appeared comparable, except for DHEA-sulfate with which zebrafish Sts appeared less efficient. While zebrafish Sts was catalytically active at 28 °C, the enzyme appeared more active at 37 °C and with similar Km values to those determined at 28 °C. Assays performed in the presence of different divalent cations showed that the activities of both zebrafish and human STSs were stimulated by Ca2+, Mg2+, and Mn2+, and inhibited by Zn+2 and Fe2+. EMATE and STX64, two known mammalian steroid sulafatase inhibitors, were shown to be capable of inhibiting the activity of zebrafish Sts. Collectively, the results obtained indicated that zebrafish Sts exhibited enzymatic characteristics comparable to the human STS, suggesting that the physiological function of STS may be conserved between zebrafish and humans.

Effects of genetic polymorphisms on the sulfation of dehydroepiandrosterone and pregnenolone by human cytosolic sulfotransferase SULT2A1.

The cytosolic sulfotransferase (SULT) SULT2A1 is known to mediate the sulfation of DHEA as well as some other hydroxysteroids such as pregnenolone. The present study was designed to investigate how genetic polymorphisms of the human SULT2A1 gene may affect the sulfation of DHEA and pregnenolone. Online databases were systematically searched to identify human SULT2A1 single nucleotide polymorphisms (SNPs). Of the 98 SULT2A1 non-synonymous coding SNPs identified, seven were selected for further investigation. Site-directed mutagenesis was used to generate cDNAs encoding these seven SULT2A1 allozymes, which were expressed in BL21 Escherichia coli cells and purified by glutathione-Sepharose affinity chromatography. Enzymatic assays revealed that purified SULT2A1 allozymes displayed differential sulfating activity toward both DHEA and pregnenolone. Kinetic analyses showed further differential catalytic efficiency and substrate affinity of the SULT2A1 allozymes, in comparison with wild-type SULT2A1. These findings provided useful information concerning the effects of genetic polymorphisms on the sulfating activity of SULT2A1 allozymes.

On the role of genetic polymorphisms in the sulfation of cholesterol by human cytosolic sulphotransferase SULT2B1b.

Sulphated cholesterol, like its unsulphated counterpart, is known to be biologically active and serves a myriad of biochemical/physiological functions. Of the 13 human cytosolic sulphotransferases (SULTs), SULT2B1b has been reported as the main enzyme responsible for the sulphation of cholesterol. As such, SULT2B1b may play the role as a key regulator of cholesterol metabolism. Variations in the sulphating activity of SULT2B1b may affect the sulphation of cholesterol and, consequently, the related physiological events. This study was designed to evaluate the impact of the genetic polymorphisms on the sulphation of cholesterol by SULT2B1b. Ten recombinant SULT2B1b allozymes were generated, expressed, and purified. Purified SULT2B1b allozymes were shown to display differential cholesterol-sulphating activities, compared with the wild-type enzyme. Kinetic studies revealed further their distinct substrate affinity and catalytic efficiency toward cholesterol. These findings showed clearly the impact of genetic polymorphisms on the cholesterol-sulphating activity of SULT2B1b allozymes, which may underscore the differential metabolism of cholesterol in individuals with different SULT2B1b genotypes.

The critical role of His48 in mouse cytosolic sulfotransferase SULT2A8 for the 7α-hydroxyl sulfation of bile acids.

Members of the cytosolic sulfotransferase (SULT) SULT2A subfamily are known to be critically involved in the homeostasis of steroids and bile acids. SULT2A8, a 7α-hydroxyl bile acid-preferring mouse SULT, has been identified as the major enzyme responsible for the mouse-specific 7-O-sulfation of bile acids. Interestingly, SULT2A8 lacks a conservative catalytic His residue at position 99th. The catalytic mechanism underlying the SULT2A8-mediated 7-O-sulfation of bile acids thus remained unclear. In this study, we performed a mutational analysis in order to gain insight into this yet-unresolved issue. Results obtained revealed two amino acid residues, His48 and Leu99, that are unique to the mouse SULT2A8, but not other SULTs, are essential for its 7-O-sulfating activity toward bile acids. These findings suggested that substitutions of two amino acids, which might have occurred during the evolution of the mouse SULT2A8 gene, endowed mouse SULT2A8 the capacity to catalyze the 7-O-sulfation of bile acids.

Radical scavenging effects of 1-naphthol, 2-naphthol, and their sulfate-conjugates.

1-Naphthol (1-Nap) and 2-naphthol (2-Nap) are phenolic isomers that may be subjected to sulfate conjugation in vivo. Phase-II sulfate conjugation of phenolic compounds is generally thought to result in their inactivation. This study aimed to investigate the antioxidative effects of 1-NapS and 2-NapS, in comparison with their unsulfated counterparts, using established free radical scavenging assays. Based on the calculated EC50 values, 1-NapS resulted in 5.60 to 7.35-times lower antioxidative activity than 1-Nap. In contrast, 2-NapS showed comparable activities as did the unsulfated 2-Nap. Collectively, the results obtained indicated that sulfate conjugation of the Nap isomers did not always result in the decrease of their antioxidant activity, and the antioxidant activity that remained appeared to depend on the position of sulfation.

Sulfation of catecholamines and serotonin by SULT1A3 allozymes.

Previous studies have demonstrated the involvement of sulfoconjugation in the metabolism of catecholamines and serotonin. The current study aimed to clarify the effects of single nucleotide polymorphisms (SNPs) of human SULT1A3 and SULT1A4 genes on the enzymatic characteristics of the sulfation of dopamine, epinephrine, norepinephrine and serotonin by SULT1A3 allozymes. Following a comprehensive search of different SULT1A3 and SULT1A4 genotypes, twelve non-synonymous (missense) coding SNPs (cSNPs) of SULT1A3/SULT1A4 were identified. cDNAs encoding the corresponding SULT1A3 allozymes, packaged in pGEX-2T vector were generated by site-directed mutagenesis. SULT1A3 allozymes were expressed, and purified. Purified SULT1A3 allozymes exhibited differential sulfating activity toward catecholamines and serotonin. Kinetic analyses demonstrated differences in both substrate affinity and catalytic efficiency of the SULT1A3 allozymes. Collectively, these findings provide useful information relevant to the differential metabolism of dopamine, epinephrine, norepinephrine and serotonin through sulfoconjugation in individuals having different SULT1A3/SULT1A4 genotypes.

Effects of Human Sulfotransferase 2A1 Genetic Polymorphisms 3 on the Sulfation of Tibolone.

BACKGROUND AND OBJECTIVES: Previous studies have demonstrated the metabolism of tibolone through sulfation, with the cytosolic sulfotransferase (SULT) SULT2A1 as the major responsible enzyme. The current study aimed to investigate how SULT2A1 genetic polymorphisms may affect the dehydroepiandrosterone (DHEA)- and tibolone-sulfating activity of SULT2A1. METHODS: Site-directed mutagenesis was employed to generate cDNAs encoding ten different SULT2A1 allozymes. Recombinant SULT2A1 allozymes were expressed in BL21 E. coli cells, and purified using glutathione-sepharose affinity chromatography. An established sulfotransferase assay was used to analyze DHEA- and tibolone-sulfating activity of the purified SULT2A1 allozymes. RESULTS: The nine human SULT2A1 allozymes plus the wild-type SULT2A1 were found to display differential sulfating activity toward DHEA and tibolone. Kinetic analysis revealed that different SULT2A1 allozymes exhibited differential substrate affinity and catalytic efficiency toward the two substrates tested. CONCLUSION: The results obtained provided useful information concerning the differential metabolism of tibolone through sulfation in individuals with different SULT2A1 genotypes.