Steroid sulfation by expressed human cytosolic sulfotransferases

doi:10.1016/0960-0760(94)90077-9

The Journal of Steroid Biochemistry and Molecular Biology

Volume 48, Issue 4, March 1994, Pages 369-375

https://doi.org/10.1016/0960-0760(94)90077-9 Get rights and content

Abstract

The human cytosolic sulfotransferases (STs), dehydroepiandrosterone sulfotransferase (DHEA-ST) and the phenol-sulfating form of phenol sulfotransferase, (P-PST), have been expressed in bacteria and used to investigate the ability of the cloned enzymes to conjugate steroids and related compounds. DHEA-ST was capable of sulfating all of the 3-hydroxysteroids, testosterone and estrogens tested as substrates. The 3-hydroxysteroids, androsterone, epiandrosterone and androstenediol, were conjugated at 50–60% of the rate of DHEA. Of the steroids tested, P-PST was capable of conjugating only the estrogens. The catechol estrogens, 2-hydroxyestradiol, 4-hydroxyestradiol and 4-hydroxyestrone, and compounds with estrogenic activity such as 17α-ethynylestradiol and trans-4-hydroxytamoxifen, were also tested as subtrates. DHEA-ST showed little or no sulfation activity with these compounds; however, all of these compounds were sulfated by P-PST. These results indicate that the expressed human STs are valuable in analyzing the overlapping substrate specificities of these enzymes and that P-PST may have an important role in the metabolism of estrogens and estrogenic compounds in human tissues.

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Conjugation of steroids and sterol compounds with a sulfonate group is a major pathway in the regulation of their activity, synthesis and excretion. Three human cytosolic sulfotransferases are highly involved in the sulfonation of sterol compounds. SULT1E1 has a low nM affinity for estrogen sulfonation and also conjugates non-aromatic steroids with a significantly lower affinity. SULT2A1 is responsible for the high levels of fetal and adult dehydroepiandrosterone (DHEA) sulfate synthesis in the adrenal gland as well as many 3α and 3ß-hydroxysteroids and bile acids. SULT2B1b is responsible for the majority of cholesterol sulfation in tissues as well as conjugating 3ß-hydroxysteroids. Although there are multiple methods for assaying cytosolic SULT activity, two relatively simple, rapid and versatile assays for steroid sulfonation are described. The first method utilizes radiolabeled substrates and organic solvent extraction to isolate the radiolabeled product from the aqueous phase. The second assay utilizes ³⁵S-3′-phosphoadenosine 5′-phosphosulfate (PAPS) to generate ³⁵S-conjugated products that are resolved by thin layer chromatography. Both assays useful in situations requiring measurement of SULT activity in a timely manner.
Structure of mouse cytosolic sulfotransferase SULT2A8 provides insight into sulfonation of 7α-hydroxyl bile acids
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Cytosolic sulfotransferases (SULTs) catalyze the transfer of a sulfonate group from the cofactor 3’-phosphoadenosine 5’-phosphosulfate to a hydroxyl (OH) containing substrate and play a critical role in the homeostasis of endogenous compounds, including hormones, neurotransmitters, and bile acids. In human, SULT2A1 sulfonates the 3-OH of bile acids; however, bile acid metabolism in mouse is dependent on a 7α-OH sulfonating SULT2A8 via unknown molecular mechanisms. In this study, the crystal structure of SULT2A8 in complex with adenosine 3’,5’-diphosphate and cholic acid was resolved at a resolution of 2.5 Å. Structural comparison with human SULT2A1 reveals different conformations of substrate binding loops. In addition, SULT2A8 possesses a unique substrate binding mode that positions the target 7α-OH of the bile acid close to the catalytic site. Furthermore, mapping of the critical residues by mutagenesis and enzyme activity assays further highlighted the importance of Lys44 and His48 for enzyme catalysis and Glu237 in loop 3 on substrate binding and stabilization. In addition, limited proteolysis and thermal shift assays suggested that the cofactor and substrates have protective roles in stabilizing SULT2A8 protein. Together, the findings unveil the structural basis of bile acid sulfonation targeting 7α-OH and shed light on the functional diversity of bile acid metabolism across species.
Δ<sup>4</sup>-3-ketosteroids as a new class of substrates for the cytosolic sulfotransferases
2017, Biochimica et Biophysica Acta - General Subjects
Cytosolic sulfotransferase (SULT)-mediated sulfation is generally known to involve the transfer of a sulfonate group from the active sulfate, 3′-phosphoadenosine 5′-phosphosulfate (PAPS), to a hydroxyl group or an amino group of a substrate compound. We report here that human SULT2A1, in addition to being able to sulfate dehydroepiandrosterone (DHEA) and other hydroxysteroids, could also catalyze the sulfation of Δ⁴-3-ketosteroids, which carry no hydroxyl groups in their chemical structure. Among a panel of Δ⁴-3-ketosteroids tested as substrates, 4-androstene-3,17-dione and progesterone were found to be sulfated by SULT2A1. Mass spectrometry analysis and structural modeling supported a reaction mechanism which involves the isomerization of Δ⁴-3-ketosteroids from the keto form to an enol form, prior to being subjected to sulfation. Results derived from this study suggested a potential role of SULT2A1 as a Δ⁴-3-ketosteroid sulfotransferase in steroid metabolism.
Human liver cytosolic sulfotransferase 2A1-dependent dehydroepiandrosterone sulfation assay by ultra-high performance liquid chromatography-tandem mass spectrometry
2016, Journal of Pharmaceutical and Biomedical Analysis
Sulfotransferase 2A1 (SULT2A1) is a major catalyst of the sulfation of dehydroepiandrosterone (DHEA) to dehydroepiandrosterone sulfate (DHEA-S) in human liver cytosol. However, there is a lack of a sensitive and fast analytical method for the human liver cytosolic SULT2A1-dependent DHEA sulfation assay. Therefore, we developed and validated an ultra-high performance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) method to quantify DHEA-S and used it to optimize the human liver cytosolic SULT2A1-dependent DHEA sulfation assay. DHEA-S and cortisol (internal standard) eluted at 2.95 and 2.75 min, respectively. Negative multiple reaction monitoring was used to quantify DHEA-S (m/z 367.3 → 97.0) and cortisol (m/z 407.2 → 331.3). No interfering peaks were observed in blank samples. The lower limit of quantification was 0.2 pmol DHEA-S and the calibration curve was linear from 0.2 to 200 pmol. The intra-day and inter-day accuracy and precision was <11.7%. DHEA-S in the quality control samples was stable at room temperature, 4 °C, and −20 °C. The cytosolic matrix (20–100 μg cytosolic protein) did not affect DHEA-S quantification. Our UPLC–MS/MS method was applied to optimize the human liver cytosolic SULT2A1-dependent DHEA sulfation assay. The optimal levels of MgCl₂ and 3′-phosphoadenosine 5′-phosphosulfate (PAPS) cofactor were 2.5 mM and 20 μM, respectively. Reducing agents, including 2-mercaptoethanol and DL-dithiothreitol, did not affect the enzyme activity. A linear relationship existed between DHEA sulfation and amount of human liver cytosol (20–200 μg cytosolic protein) or incubation time (5–30 min). This UPLC–MS/MS approach is safer, easier, and faster than existing radiometric-based sulfotransferase enzyme assays, and it is the first UPLC–MS/MS method for determining SULT2A1-dependent DHEA sulfation in human liver cytosol.
Celecoxib influences steroid sulfonation catalyzed by human recombinant sulfotransferase 2A1
2015, Journal of Steroid Biochemistry and Molecular Biology
Citation Excerpt :
To the contrary, computational analyses of protein–ligand docking suggested that celecoxib occupied the same binding region as 17β-E2 and modulated SULT2A1-catalyzed 17β-E2 sulfonation by inducing a conformational change that favored docking of 17β-E2 into an alternative binding region such that the 17β-hydroxy group was sulfonated in preference to the 3-OH group [13]. SULT1E1 and SULT1A1 have been shown to sulfonate 17β-E2 with higher affinity and capacity than SULT2A1 [14,15]. SULT1E1 particularly has very high affinity for 17β-E2 and is thought to play the most important role in its sulfonation at physiological low nM concentrations [16].
Celecoxib has been reported to switch the human SULT2A1-catalyzed sulfonation of 17β-estradiol (17β-E2) from the 3- to the 17-position. The effects of celecoxib on the sulfonation of selected steroids catalyzed by human SULT2A1 were assessed through in vitro and in silico studies. Celecoxib inhibited SULT2A1-catalyzed sulfonation of dehydroepiandrosterone (DHEA), androst-5-ene-3β, 17β-diol (AD), testosterone (T) and epitestosterone (Epi-T) in a concentration-dependent manner. Low μM concentrations of celecoxib strikingly enhanced the formation of the 17-sulfates of 6-dehydroestradiol (6D-E2), 17β-dihydroequilenin (17β-Eqn), 17β-dihydroequilin (17β-Eq), and 9-dehydroestradiol (9D-E2) as well as the overall rate of sulfonation. For 6D-E2, 9D-E2 and 17β-Eqn, celecoxib inhibited 3-sulfonation, however 3-sulfonation of 17β-Eq was stimulated at celecoxib concentrations below 40 μM. Ligand docking studies in silico suggest that celecoxib binds in the substrate-binding site of SULT2A1 in a manner that prohibits the usual binding of substrates but facilitates, for appropriately shaped substrates, a binding mode that favors 17-sulfonation.
Estrogen sulfotransferase (SULT1E1): Its molecular regulation, polymorphisms, and clinical perspectives
2021, Journal of Personalized Medicine

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Steroid sulfation by expressed human cytosolic sulfotransferases

Abstract

Archs Biochem. Biophys.

Biochem. Pharmac.

Biochim. Biophys. Acta

J. Steroid Biochem.

J. Steroid Biochem.

Trends Pharmac. Sci.

Trends Pharmac. Sci.

J. Steroid Biochem. Molec. Biol.

The Biologic Role for Dehydroepiandrosterone

The effects of oral dehydroepiandrosterone on endocrin-metabolic parameters in postmenopausal women

J. Clin Endocr. Metab.

Further studies on the relationship between C19 and C21 steriod synthesis in the human adrenal gland

J. Endocr

Sulphation of Bile Acids

Purification and characterization of human liver dehydroepiandrosterone sulfotransferase

Biochem. J.