المؤلفون: Cook, Ian¹, Ting Wang¹, Leyh, Thomas S.¹ tom.leyh@einstein.yu.edu

المصدر: Biochemistry. 10/06/2015, Vol. 54 Issue 39, p6114-6122. 9p.

مصطلحات موضوعية: *SULFOTRANSFERASES, *BIOCHEMICAL substrates, *METABOLITES, *XENOBIOTICS, *HYDROXYL group, *MOLECULAR dynamics

مستخلص: The human cytosolic sulfotransferases (SULTs) regulate hundreds, perhaps thousands, of small molecule metabolites and xenobiotics via transfer of a sulfuryl moiety (-SO3) from PAPS (3′-phosphoadenosine 5′-phosphosulfate) to the hydroxyls and primary amines of the recipients. In liver, where it is abundant, SULT1A1 engages in modifying metabolites and neutralizing toxins. The specificity of 1A1 is the broadest of any SULT, and understanding its selectivity is fundamental to understanding its biology. Here, for the first time, we show that SULT1A1 substrates separate naturally into two classes: those whose affinities are either enhanced ∼20-fold (positive synergy) or unaffected (neutral synergy) by the presence of a saturating nucleotide. kcat for the positive-synergy substrates is shown to be ∼100-fold greater than that of neutral-synergy compounds; consequently, the catalytic efficiency (kcat/Km) is approximately 3 orders of magnitude greater for the positive-synergy species. All-atom dynamics modeling suggests a molecular mechanism for these observations in which the binding of only positive-synergy compounds causes two phenylalanine residues (F81 and 84) to reposition and “sandwich” the phenolic moiety of the substrates, thus enhancing substrate affinity and positioning the nucleophilic oxygen for attack. Molecular dynamics movies reveal that the neutral-synergy compounds “wander” about the active site, infrequently achieving a reactive position. In-depth analysis of select point mutants strongly supports the model and provides an intimate view of the interdependent catalytic functions of subsections of the active site. [ABSTRACT FROM AUTHOR]

المؤلفون: Kranendonk, Michel¹, Alves, Mónica¹, Antunes, Pedro¹, Rueff, José¹

المصدر: Chemical Research in Toxicology. Nov2014, Vol. 27 Issue 11, p1967-1971. 5p.

مصطلحات موضوعية: *SULFOTRANSFERASES, *MUTAGENICITY testing, *NEVIRAPINE, *ANTI-HIV agents, *DRUG efficacy, *METABOLITES

مستخلص: Nevirapine(NVP) is a frequently used anti-HIV drug. Despite itsefficacy, NVP has been associated with serious skin and liver injuriesin exposed patients and with increased incidences of hepatoneoplasiasin rodents. Current evidence supports the involvement of reactivemetabolites in the skin and liver toxicities of NVP, formed by cytochromeP450-mediated oxidations and/or subsequent phase II sulfonation. However,to date, standard in vitrogenotoxicity tests haveprovided no evidence that NVP is either mutagenic or clastogenic.The human sulfotransferase 1A1-dependent mutagenicity of 12-hydroxy-NVP,one of the major metabolites of NVP, is demonstrated here. [ABSTRACT FROM AUTHOR]

المؤلفون: Ting Wang¹, Cook, Ian¹, Leyh, Thomas S.¹ tom.leyh@einstein.yu.edu

المصدر: Biochemistry. 11/11/2014, Vol. 53 Issue 44, p6893-6900. 8p.

مصطلحات موضوعية: *SULFOTRANSFERASES, *ADENOSINES, *NUCLEOTIDES, *ENZYMES, *METABOLITES, *BIOMOLECULES, *BIOCHEMISTRY

مستخلص: Human cytosolic sulfotransferases (SULTs) regulate the activities of thousands of small molecules-metabolites, drugs, and other xenobiotics-via the transfer of the sulfuryl moiety (-SO3) from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the hydroxyls and primary amines of acceptors. SULT1A1 is the most abundant SULT in liver and has the broadest substrate spectrum of any SULT. Here we present the discovery of a new form of SULT1A1 allosteric regulation that modulates the catalytic efficiency of the enzyme over a 130-fold dynamic range. The molecular basis of the regulation is explored in detail and is shown to be rooted in an energetic coupling between the active-site caps of adjacent subunits in the SULT1A1 dimer. The first nucleotide to bind causes closure of the cap to which it is bound and at the same time stabilizes the cap in the adjacent subunit in the open position. Binding of the second nucleotide causes both caps to open. Cap closure sterically controls active-site access of the nucleotide and acceptor; consequently, the structural changes in the cap that occur as a function of nucleotide occupancy lead to changes in the substrate affinities and turnover of the enzyme. PAPS levels in tissues from a variety of organs suggest that the catalytic efficiency of the enzyme varies across tissues over the full 130-fold range and that efficiency is greatest in those tissues that experience the greatest xenobiotic "load". [ABSTRACT FROM AUTHOR]

المؤلفون: Bendadani, Carolin¹, Meinl, Walter¹, Monien, Bernhard¹, Dobbernack, Gisela¹, Florian, Simone¹, Engst, Wolfram¹, Nolden, Tobias¹, Himmelbauer, Heinz¹, Glatt, Hansruedi¹

المصدر: Chemical Research in Toxicology. Jun2014, Vol. 27 Issue 6, p1060-1069. 10p.

مصطلحات موضوعية: *SULFOTRANSFERASES, *BIOTRANSFORMATION (Metabolism), *DNA adducts, *ENZYMES, *POLYCYCLIC aromatic hydrocarbons, *DEOXYGUANOSINE

مستخلص: 1-Methylpyrene, a carcinogenic polycyclicaromatic hydrocarbon,forms benzylic DNA adducts, in particular N2-(1-methylpyrenyl)-2′-deoxyguanosine, in mice and rats. Itis bioactivated via 1-hydroxymethylpyrene (1-HMP) to electrophilic1-sulfooxymethylpyrene (1-SMP). In this study, we explored the roleof individual mouse sulfotransferase (SULT) forms in this activation.First, we showed that all nine mouse SULTs tested were able to activate1-HMP to a mutagen in the his–Salmonellatyphimuriumreversion test. Some activation was even observedwith Sult2a3 and Sult5a1, orphan forms for which no substrates wereidentified hitherto. Subsequently, we used cytosolic preparationsfrom tissues of four mouse lines (wild-type, Sult1a1–, Sult1d1–, and transgenic for human SULT1A1/2)for the activation of 1-HMP in the mutagenicity assay. The most prominentimpacts of the genetic SULT status were 96% decrease in hepatic activationby Sult1a1 knockout, 99% decrease in renal activation by Sult1d1 knockout,and 100-fold increase in pulmonary activation by transgenic humanSULT1A1/2. Finally, we treated the various mouse lines with 1-HMP(19.3 mg/kg, intraperitoneally), and then determined 1-SMP levelsin plasma and DNA adducts in tissues. Transgenic human SULT1A1/2 stronglyenhanced 1-SMP plasma levels and DNA adduct formation in the liver,lung, heart, and kidney but not in the colon. Sult1a1 and Sult1d1knockout reduced plasma 1-SMP levels as well as DNA adduct formationin some tissues (strongest effects: 97% decrease in 1-SMP and 89%decrease in hepatic adducts in Sult1a1–mice). Theadduct levels detected in various tissues did not accurately reflectthe activation capacity of these tissues determined in vitro, probably due to the distribution of the reactive metabolite 1-SMPvia the circulation. In conclusion, we demonstrated that many mouseSULT forms are able to activate 1-HMP. In vivo, weverified a prominent role of Sult1a1 in hepatic and renal adduct formationand a smaller but unambiguous role of Sult1d1, and demonstrated thestrong impact of transgenic human SULT1A1/2. [ABSTRACT FROM AUTHOR]

المؤلفون: Malojčić, Goran¹ goran@malojcic@alumni.ethz.ch, Owen, Robin L.², Glockshuber, Rudi¹

المصدر: Biochemistry. 3/25/2014, Vol. 53 Issue 11, p1870-1877. 8p.

مصطلحات موضوعية: *SULFOTRANSFERASES, *ARYLSULFONATES, *ESCHERICHIA coli, *CRYSTAL structure, *STOICHIOMETRY

مستخلص: Bacterial aryl sulfotransferases (ASSTs) catalyze sulfotransfer from a phenolic sulfate to a phenol. These enzymes are frequently found in pathogens and upregulated during infection. Their mechanistic understanding is very limited, and their natural substrates are unknown. Here, the crystal structures of Escherichia coli CFT073 ASST trapped in its presulfurylation state with model donor substrates bound in the active site are reported, which reveal the molecular interactions governing substrate recognition. Furthermore, spectroscopic titrations with donor substrates and sulfurylation kinetics of ASST illustrate that this enzyme binds substrates in a 1:1 stoichiometry and that the active sites of the ASST homooligomer act independently. Mass spectrometry and crystallographic experiments of ASST incubated with human urine demonstrate that urine contains a sulfuryl donor substrate. In addition, we examined the capability of the two paralogous dithiol oxidases present in uropathogenic E. coli CFT073, DsbA, and the ASST-specific enzyme DsbL, to introduce the single, conserved disulfide bond into ASST. We show that DsbA and DsbL introduce the disulfide bond into unfolded ASST at similar rates. Hence, a chaperone effect of DsbL, not present in DsbA, appears to be responsible for the dependence of efficient ASST folding on DsbL in vivo. The conservation of paralogous dithiol oxidases with different substrate specificities in certain bacterial strains may therefore be a consequence of the complex folding pathways of their substrate proteins. [ABSTRACT FROM AUTHOR]

المؤلفون: Butt, Craig M.¹, Stapleton, Heather M.¹

المصدر: Chemical Research in Toxicology. Nov2013, Vol. 26 Issue 11, p1692-1702. 11p.

مصطلحات موضوعية: *THYROID hormones, *SULFOTRANSFERASES, *BROMINATION, *FIREPROOFING agents, *PHENOLS, *ENDOCRINE disruptors

مستخلص: Many halogenated organic contaminants(HOCs) are considered endocrinedisruptors and affect the hypothalamic–pituitary–thyroidaxis, often by interfering with circulating levels of thyroid hormones(THs). We investigated one potential mechanism for TH disruption,inhibition of sulfotransferase activity. One of the primary rolesof TH sulfation is to support the regulation of biologically activeT3 through the formation of inactive THs. We investigated TH sulfotransferaseinhibition by 14 hydroxylated polybrominated diphenyl ethers (OH BDEs),BDE 47, triclosan, and fluorinated, chlorinated, brominated, and iodinatedanalogues of 2,4,6-trihalogenated phenol and bisphenol A (BPA). Anew mass spectrometry-based method was also developed to measure theformation rates of 3,3′-T2 sulfate (3,3′-T2S). Usingpooled human liver cytosol, we investigated the influence of theseHOCs on the sulfation of 3,3′-T2, a major substrate for THsulfation. For the formation of 3,3′-T2S, the Michaelis constant(Km) was 1070 ± 120 nM and the Vmaxwas 153 ± 6.6 pmol min–1(mg of protein)−1. All chemicals investigatedinhibited sulfotransferase activity with the exception of BDE 47.The 2,4,6-trihalogenated phenols were the most potent inhibitors followedby the OH BDEs and then halogenated BPAs. The IC50valuesfor the OH BDEs were primarily in the low nanomolar range, which maybe environmentally relevant. In silicomolecularmodeling techniques were also used to simulate the binding of OH BDEto SULT1A1. This study suggests that some HOCs, including antimicrobialchemicals and metabolites of flame retardants, may interfere withTH regulation through inhibition of sulfotransferase activity. [ABSTRACT FROM AUTHOR]

المؤلفون: Cook, Ian¹, Ting Wang¹, Almo, Steven C.², Jungwook Kim², Falany, Charles N.³, Leyh, Thomas S.^1,3 leyh@einstein.yu.edu

المصدر: Biochemistry. 1/15/2013, Vol. 52 Issue 2, p415-424. 10p.

مصطلحات موضوعية: *SULFOTRANSFERASES, *HYDROXY acids, *METABOLITES, *MOLECULAR dynamics, *XENOBIOTICS, *SULFURYL chloride, *CYTOSOL, *ISOMERIZATION

مستخلص: Human cytosolic sulfotransferases (SULTs) transfer the sulfuryl moiety (-SO3) from activated sulfate [3'-phosphoadenosine 5'-phosphosulfate (PAPS)] to the hydroxyls and primary amines of numerous metabolites, drugs, and xenobiotics. Receipt of the sulfuryl group often radically alters acceptor-target interactions. How these enzymes select particular substrates from the hundreds of candidates in a complex cytosol remains an important question. Recent work reveals PAPS binding causes SULT2A1 to undergo an isomerization that controls selectivity by constricting the opening through which acceptors must pass to enter the active site. The enzyme maintains an affinity for large substrates by isomerizing between the open and closed states with nucleotide bound. Here, the molecular basis of the nucleotide-induced closure is explored in equilibrium and nonequilibrium molecular dynamics simulations. The simulations predict that the active-site "cap," which covers both the nucleotide and acceptor binding sites, opens and closes in response to nucleotide. The cap subdivides into nucleotide and acceptor halves whose motions, while coupled, exhibit an independence that can explain the isomerization. In silico weakening of electrostatic interactions between the cap and base of the active site causes the acceptor half of the cap to open and close while the nucleotide lid remains shut. Simulations predict that SULT1A1, the most abundant SULT in human liver, will utilize a similar selection mechanism. This prediction is tested using fulvestrant, an anti-estrogen too large to pass through the closed pore, and estradiol, which is not restricted by closure. Equilibrium and pre-steady-state binding studies confirm that SULT1A1 undergoes a nucleotide-induced isomerzation that controls substrate selection. [ABSTRACT FROM AUTHOR]

المؤلفون: Cook, Ian¹, Ting Wang¹, Falany, Charles N.², Leyh, Thomas S.¹ leyh@einstein.yu.edu

المصدر: Biochemistry. 7/17/2012, Vol. 51 Issue 28, p5674-5683. 10p.

مصطلحات موضوعية: *SULFOTRANSFERASES, *SULFATES, *RALOXIFENE, *ISOMERIZATION, *DEHYDROEPIANDROSTERONE

مستخلص: Human SULT2AI is one of two predominant sulfotransferases in liver and catalyzes transfer of the sulfuryl moiety (-SO3) from activated sulfate (PAPS, 3'-phosphoadenosine 5-phosphosulfate) to hundreds of acceptors (metabolites and xenobiotics). Sulfation recodes the biologic activity of acceptors by altering their receptor interactions. The molecular basis on which these enzymes select and sulfonate specific acceptors from complex mixtures of competitors in vivo is a long-standing issue in the SULT field. Raloxifene, a synthetic steroid used in the prevention of osteoporosis, and dehydroepiandrosterone (DHEA), a ubiquitous steroid precusor, are reported to be sulfated efficiently by SULT2AI in vitro, yet unlike DHEA, raloxifene is not sulfated in vivo. This selectivity was explored in initial rate and equilibrium binding studies that demonstrate pronounced binding antisynergy (21-fold) between PAPS and raloxifene, but not DHEA. Analysis of crystal structures suggests that PAP binding restricts access to the acceptor-binding pocket by restructuring a nine-residue segment of the pocket edge that constricts the active site opening, or "pore", that sieves substrates on the basis of their geometries. In silico docking predicts that raloxifene, which is considerably larger than DHEA, can bind only to the unliganded (open) enzyme, whereas DHEA binds both the open and dosed forms. The predictions of these structures with regard to substrate binding are tested using equilibrium and pre-steady-state ligand binding studies, and the results confirm that a nudeotide-driven isomerization controls access to the acceptor-binding pocket and plays an important role in substrate selection by SULT2AI and possibly other sulfotransferases. [ABSTRACT FROM AUTHOR]

المؤلفون: Danan, Lieza M.¹, Yu, Zhihao¹, Ludden, Peter J.¹, Jia, Weitao¹, Moore, Kevin L.^2,3,4, Leary, Julie A.¹ jaleary@ucdavis.edu

المصدر: Journal of the American Society for Mass Spectrometry. Sep2010, Vol. 21 Issue 9, p1633-1642. 10p.

مصطلحات موضوعية: *CATALYSIS, *SULFOTRANSFERASES, *PROTEINS, *PEPTIDES, *TYROSINE, *LIQUID chromatography, *MASS spectrometry

مستخلص: Human tyrosylprotein sulfotransferases catalyze the transfer of a sulfuryl moiety from the universal sulfate donor PAPS to the hydroxyl substituent of tyrosine residues in proteins and peptides to yield tyrosine sulfated products and PAP. Tyrosine sulfation occurs in the trans-Golgi network, affecting an estimated 1% of the tyrosine residues in all secreted and membrane-bound proteins in higher order eukaryotes. In this study, an effective LC-MS-based TPST kinetics assay was developed and utilized to measure the kinetic properties of human TPST-2 and investigate its catalytic mechanism when G protein-coupled CC-chemokine receptor 8 (CCR8) peptides were used as acceptor substrates. Through initial rate kinetics, product inhibition studies, and radioactive-labeling experiments, our data strongly suggest a two-site ping-pong model for TPST-2 action. In this mechanistic model, the enzyme allows independent binding of substrates to two distinct sites, and involves the formation of a sulfated enzyme covalent intermediate. Some insights on the important amino acid residues at the catalytic site of TPST-2 and its covalent intermediate are also presented. To our knowledge, this is the first detailed study of the reaction kinetics and mechanism reported for human TPST-2 or any other Golgi-resident sulfotransferase. [ABSTRACT FROM AUTHOR]

المؤلفون: Sun, Meihao¹, Lcyh, Thomas S.² leyh@accom.yu.edu

المصدر: Biochemistry. 6/15/2010, Vol. 49 Issue 23, p4779-4785. 7p.

مصطلحات موضوعية: *SULFOTRANSFERASES, *ESTROGEN receptors, *ENZYMES, *REACTION mechanisms (Chemistry), *CHEMICAL reactions, *DIMERS, *REACTIVITY (Chemistry)

مستخلص: The affinity of 17β-estradiol (E2) for the estrogen receptor is weakened beyond the point of physiological relevance by the transfer of the sulfuryl moiety (-SO3) from PAPS (3'-phosphoadenosine 5'-phosphosulfate) to the 3'-hydroxyl of E2. The mechanism of this transfer reaction, catalyzed by estrogen sulfotransferase (EST). is investigated here in detail. The enzyme (a dimer of identical protomers) presents a clear example of half-sites reactivity—only one of the subunits of the dimer produces product during the catalytic cycle. This is the first example of half-sites reactivity in the sulfotransferase family. A burst of product, with an amplitude that corresponds to one-half of the available active sites, reveals that the mechanism is rate-limited by product release. The equilibrium constant governing interconversion of the substrate (E ∙ PAPS ∙ E2) and product (E ∙ PAPS ∙ E2) central complexes was determined and is strongly biased toward product (Keq int ∼49). Slow product release allows the interconversion of the central complexes to approach equilibrium, with the result that Keq int becomes nearly linearly coupled to Km and contributes a factor of -30 to the steady-state affinity of the enzyme for substrate. Typical of its family, estrogen sulfotransferase is partially keat-inhibited by its acceptor substrate, E2. This inhibition does not influence the burst kinetics and thus occurs after formation of the product central complex, a finding consistent with the slow escape of PAP from the nonreactive E ∙ PAPS ∙ E2 complex. [ABSTRACT FROM AUTHOR]