Human cytosolic steroid sulfotransferases: Versatile and rapid activity assays.

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.

N-terminal domain of tyrosyl-DNA phosphodiesterase I regulates topoisomerase I-induced toxicity in cells.

Tyrosyl-DNA phosphodiesterase I (Tdp1) hydrolyzes phosphodiester-linked adducts from both ends of DNA. This includes the topoisomerase I (TOP1)-DNA covalent reaction intermediate that is the target of the camptothecin class of chemotherapeutics. Tdp1 two-step catalysis is centered on the formation of a Tdp1-DNA covalent complex (Tdp1cc) using two catalytic histidines. Here, we examined the role of the understudied, structurally undefined, and poorly conserved N-terminal domain (NTD) of Tdp1 in context of full-length protein in its ability to remove TOP1cc in cells. Using toxic Tdp1 mutants, we observed that the NTD is critical for Tdp1's ability to remove TOP1-DNA adducts in yeast. Full-length and N-terminal truncated Tdp1 mutants showed similar expression levels and cellular distribution yet an inversed TOP1-dependent toxicity. Single turnover catalysis was significantly different between full-length and truncated catalytic mutants but not wild-type enzyme, suggesting that Tdp1 mutants depend on the NTD for catalysis. These observations suggest that the NTD plays a critical role in the regulation of Tdp1 activity and interaction with protein-DNA adducts such as TOP1cc in cells. We propose that the NTD is a regulatory domain and coordinates stabilization of the DNA-adducted end within the catalytic pocket to access the phosphodiester linkage for hydrolysis.

Sulfotransferase 4A1 activity facilitates sulfate-dependent cellular protection to oxidative stress.

Sulfotransferase 4A1 (SULT4A1) is an orphan member of the cytosolic SULT superfamily that contains enzymes that catalyze the sulfonation of hydrophobic drugs and hormones. SULT4A1 has been assessed through all classical SULT approaches yet no SULT activity has been reported. To ascertain SULT4A1 function and activity, we utilized Saccharomyces cerevisiae as a model system, which exhibits no endogenous SULT activity nor possesses SULT-related genes. We observed that ectopic SULT4A1 expression in yeast displays similar subcellular localization as reported in mouse neurons and observed that SULT4A1 is associated with the outer mitochondria membrane. SULT4A1 expression stimulates colony formation and protects these cells from hydrogen peroxide and metabolism-associated oxidative stress. These SULT4A1-mediated phenotypes are dependent on extracellular sulfate that is converted in yeast to PAPS, the universal sulfonate donor for SULT activity. Thus, heterologous SULT4A1 expression in yeast is correctly distributed and functional, and SULT4A1 antioxidant activity is sulfate dependent supporting the concept that SULT4A1 has sulfate-associated activity.

Restoration of CTSD (cathepsin D) and lysosomal function in stroke is neuroprotective.

Stroke is a leading cause of death and disability. The pathophysiological mechanisms associated with stroke are very complex and not fully understood. Lysosomal function has a vital physiological function in the maintenance of cellular homeostasis. In neurons, CTSD (cathepsin D) is an essential protease involved in the regulation of proteolytic activity of the lysosomes. Loss of CTSD leads to lysosomal dysfunction and accumulation of different cellular proteins implicated in neurodegenerative diseases. In cerebral ischemia, the role of CTSD and lysosomal function is not clearly defined. We used oxygen-glucose deprivation (OGD) in mouse cortical neurons and the middle cerebral artery occlusion (MCAO) model of stroke to assess the role of CTSD in stroke pathophysiology. Our results show a time-dependent decrease in CTSD protein levels and activity in the mouse brain after stroke and neurons following OGD, with concurrent defects in lysosomal function. We found that shRNA-mediated knockdown of CTSD in neurons is sufficient to cause lysosomal dysfunction. CTSD knockdown further aggravates lysosomal dysfunction and cell death in OGD-exposed neurons. Restoration of CTSD protein levels via lentiviral transduction increases CTSD activity in neurons and, thus, renders resistance to OGD-mediated defects in lysosomal function and cell death. This study indicates that CTSD-dependent lysosomal function is critical for maintaining neuronal survival in cerebral ischemia; thus, strategies focused on maintaining CTSD function in neurons are potentially novel therapeutic approaches to prevent neuronal death in stroke.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AD: Alzheimer disease; ALS: amyotrophic lateral sclerosis; CQ: chloroquine; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; FTD: frontotemporal dementia, HD: Huntington disease; LAMP1: lysosomal associated membrane protein 1; LSD: lysosomal storage disease; MCAO: middle cerebral artery occlusion; OGD: oxygen glucose deprivation; OGR: oxygen glucose resupply; PD: Parkinson disease; SQSMT1: sequestosome 1; TCA: trichloroacetic acid; TTC: triphenyl tetrazolium chloride.

Evolutionary Analysis of Bile Acid-Conjugating Enzymes Reveals a Complex Duplication and Reciprocal Loss History.

To fulfill their physiological functions, bile acids are conjugated with amino acids. In humans, conjugation is catalyzed by bile acid coenzyme A: amino acid N-acyltransferase (BAAT), an enzyme with a highly conserved catalytic triad in its active site. Interestingly, the conjugated amino acids are highly variable among mammals, with some species conjugating bile acids with both glycine and taurine, whereas others conjugate only taurine. The genetic origin of these bile acid conjugation differences is unknown. Here, we tested whether mutations in BAAT's catalytic triad could explain bile acid conjugation differences. Our comparative analysis of 118 mammals first revealed that the ancestor of placental mammals and marsupials possessed two genes, BAAT and BAATP1, that arose by a tandem duplication. This duplication was followed by numerous gene losses, including BAATP1 in humans. Losses of either BAAT or BAATP1 largely happened in a reciprocal fashion, suggesting that a single conjugating enzyme is generally sufficient for mammals. In intact BAAT and BAATP1 genes, we observed multiple changes in the catalytic triad between Cys and Ser residues. Surprisingly, although mutagenesis experiments with the human enzyme have shown that replacing Cys for Ser greatly diminishes the glycine-conjugating ability, across mammals we found that this residue provides little power in predicting the experimentally measured amino acids that are conjugated with bile acids. This suggests that the mechanism of BAAT's enzymatic function is incompletely understood, despite relying on a classic catalytic triad. More generally, our evolutionary analysis indicates that results of mutagenesis experiments may not easily be extrapolatable to other species.

SULT4A1 Protects Against Oxidative-Stress Induced Mitochondrial Dysfunction in Neuronal Cells.

Sulfotransferase 4A1 (SULT4A1), a member of cytosolic sulfotransferases (SULT), is exclusively expressed in neurons with no known function. Severe phenotype and early postnatal death in SULT4A1 knockout mice revealed that SULT4A1 is an essential neuronal protein. Localization of SULT4A1 in different cytosolic compartments, including mitochondria, suggests multiple roles for this protein. We observed that knockdown of SULT4A1 results in the accumulation of reactive oxygen species in primary cortical neurons, suggesting a potential role of SULT4A1 in regulating redox homeostasis. Expression of SULT4A1 in the human neuroblastoma SH-SY5Y cells revealed a defused but nonuniform staining pattern in the cytoplasm, with increased density around mitochondria. Subcellular fractionation of SULT4A1 expressing SH-SY5Y cells confirms the presence of SULT4A1 in mitochondrial fractions. SULT4A1 expressing cells display significant protection against H2O2-mediated defects in mitochondrial function and loss of mitochondrial membrane potential. Expression of SULT4A1 in SH-SY5Y cells also protects against H2O2-induced cell death. These data indicate that SULT4A1 protects mitochondria against oxidative damage and may serve as a potential pharmacological target in neural diseases involving mitochondrial dysfunction and oxidative stress. SIGNIFICANCE STATEMENT: Studies on SULT4A1 knockout mice suggest that SULT4A1 plays a vital role in neuronal function and survival via yet undefined mechanisms. Our data demonstrate that depletion of SULT4A1 induces oxidative stress in neurons and expression of SULT4A1 in SH-SY5Y cells protects against oxidative-stress-induced mitochondrial dysfunction and cell death. These results suggest that SULT4A1 may have a crucial protective function against mitochondrial dysfunction and oxidative stress, and may serve a potential therapeutic target in different neurological diseases involving mitochondrial dysfunction and oxidative stress.

Generation and Characterization of SULT4A1 Mutant Mouse Models.

Sulfotransferase 4A1 (SULT4A1) belongs to the cytosolic sulfotransferase (SULT) superfamily of enzymes that catalyze sulfonation reactions with a variety of endogenous and exogenous substrates. Of the SULTs, SULT4A1 was shown to have the highest sequence homology between vertebrate species, yet no known function or enzymatic activity has been identified for this orphan SULT. To better understand SULT4A1 function in mammalian brain, two mutant SULT4A1 mouse strains were generated utilizing clustered regulatory interspaced short palindromic repeats (CRISPR)-content-addressable storage (Cas) 9 technology. The first strain possessed a 28-base pair (bp) deletion (Δ28) in exon 1 that resulted in a frameshift mutation with premature termination. The second strain possessed a 12-bp in-frame deletion (Δ12) immediately preceding an active site histidine111 common to the SULT family. Homozygous pups of both strains present with severe and progressive neurologic symptoms, including tremor, absence seizures, abnormal gait, ataxia, decreased weight gain compared with littermates, and death around postnatal days 21-25. SULT4A1 immunostaining was decreased in brains of heterozygous pups and not detectable in homozygous pups of both SULT4A1 mutants. SULT4A1 localization in subcellular fractions of mouse brain showed SULT4A1 associated with mitochondrial, cytosolic, and microsomal fractions, a novel localization pattern for SULTs. Finally, primary cortical neurons derived from embryonic (E15) CD-1 mice expressed high levels of SULT4A1 throughout the cell except in nuclei. Taken together, SULT4A1 appears to be an essential neuronal protein required for normal brain function, at least in mammals. Mouse models will be valuable in future studies to investigate the regulation and functions of SULT4A1 in the mammalian brain.

A high frequency missense SULT1B1 allelic variant (L145V) selectively expressed in African descendants exhibits altered kinetic properties.

1. Human cytosolic sulfotransferase 1B1 (SULT1B1) sulfates small phenolic compounds and bioactivates polycyclic aromatic hydrocarbons. To date, no SULT1B1 allelic variants have been well-characterized. 2. While cloning SULT1B1 from human endometrial specimens, an allelic variant resulting in valine instead of leucine at the 145th amino acid position (L145V) was detected. NCBI reported this alteration as the highest frequency SULT1B1 allelic variant. 3. L145V frequency comprised 9% of 37 mixed-population human patients and was specific to African Americans with an allelic frequency of 25%. Structurally, replacement of leucine with valine potentially destabilizes a conserved helix (α8) that forms the "floor" of both the substrate and PAPS binding domains. This destabilization results in altered kinetic properties including a four-fold decrease in affinity for PAP (3', 5'-diphosphoadenosine). Kms for 3'-phosphoadenosine- 5'-phosphosulfate (PAPS) are similar; however, maximal turnover rate of the variant isoform (0.86 pmol/(min*µg)) is slower than wild-type (WT) SULT1B1 (1.26 pmol/(min*µg)). The L145V variant also displays altered kinetics toward small phenolic substrates, including a diminished p-nitrophenol Km and increased susceptibility to 1-naphthol substrate inhibition. 4. No significant correlation between genotype and prostate or colorectal cancer was observed in patients; however, the variant isoform could underlie specific pathologies in sub-Saharan African carriers.

Expression, purification and characterization of human cytosolic sulfotransferase (SULT) 1C4.

Human cytosolic sulfotransferase 1C4 (hSULT1C4) is a dimeric Phase II drug-metabolizing enzyme primarily expressed in the developing fetus. SULTs facilitate the transfer of a hydrophilic sulfonate moiety from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) onto an acceptor substrate altering the substrate's biological activity and increasing the compound's water solubility. While several of the hSULTs' endogenous and xenobiotic substrates have been identified, the physiological function of hSULT1C4 remains unknown. The fetal expression of hSULT1C4 leads to the hypothesis that the function of this enzyme may be to regulate metabolic and hormonal signaling molecules, such as estrogenic compounds, that may be generated or consumed by the mother during fetal development. Human SULT1C4 has previously been shown to sulfonate estrogenic compounds, such as catechol estrogens; therefore, this study focused on the expression and purification of hSULT1C4 in order to further characterize this enzyme's sulfonation of estrogenic compounds. Molecular modeling of the enzyme's native properties helped to establish a novel purification protocol for hSULT1C4. The optimal activity assay conditions for hSULT1C4 were determined to be pH 7.4 at 37°C for up to 10 min. Kinetic analysis revealed the enzyme's reduced affinity for PAPS compared to PAP. Human SULT1C4 sulfonated all the estrogenic compounds tested, including dietary flavonoids and environmental estrogens; however, the enzyme has a higher affinity for sulfonation of flavonoids. These results suggest hSULT1C4 could be metabolizing and regulating hormone signaling pathways during human fetal development.

An engineered heterodimeric model to investigate SULT1B1 dependence on intersubunit communication.

Cytosolic sulfotransferases (SULTs) biotransform small molecules to polar sulfate esters as a means to alter their activities within the body. Understanding the molecular mechanism by which the SULTs perform their function is important for optimizing future therapeutic applications. Recent evidence suggests each SULT isoform acts by a half-site reaction (HSR) mechanism, in which a single SULT dimer subunit is active at any given time. HSR requires communication through the highly conserved KxxxTVxxxE dimerization motif. In this investigation, we sought to test the intersubunit interactions of SULT1B1 as it relates to enzyme activity. We generated two populations of SULT1B1 isoforms that efficiently heterodimerize upon mixing by targeted point mutation of the KxxxTVxxxE motif to KxxxTVxxxK or ExxxTVxxxE. The heterodimer exhibited wildtype-like activity with regard to native size, thermal integrity, PAP affinity, and PAPS Km, therefore serving as a valid model for investigating SULT1B1 dimer subunit interactions. The approach granted control over each independent subunit, permitting mutation of the critical 3'-phosphoadenosine 5'-phosphosulfate (PAPS) binding residue Arg258 and/or the catalytic base His109 in a single subunit of the dimer. Substitution of the dysfunctional subunits for fully active subunits yielded dimeric SULT1B1 with 50% the activity of the fully competent dimer, suggesting SULT1B1 intersubunit communication does not significantly contribute to the isoform's activity. These results are a testament to the unique properties of individual SULT isoforms. The dimerization system described in this manuscript can be used to study subunit interactions in other SULT isoforms as well as proteins in other families.

Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificity.

Bile acid CoA:amino acid N-acyltransferase (BAAT) is the terminal enzyme in the synthesis of bile salts from cholesterol and catalyzes the conjugation of taurine or glycine to bile acid CoA thioesters to form bile acid N-acylamidates. BAAT has a dual localization to the cytosol and peroxisomes, possibly due to an inefficient carboxy-terminal peroxisomal targeting signal (PTS), -serine-glutamine-leucine (-SQL). Mutational analysis was used to define the role of the carboxy terminus in peroxisomal localization and kinetic activity. Amidation activity of BAAT and BAAT lacking the final two amino acids (AAs) (BAAT-S) were similar, whereas the activity of BAAT with a canonical PTS sequence (BAAT-SKL) was increased >2.5-fold. Kinetic analysis of BAAT and BAAT-SKL showed that BAAT-SKL had a lower Km for taurine and glycine as well as a greater Vmax There was no difference in the affinity for cholyl-CoA. In contrast to BAAT, BAAT-SKL forms bile acid N-acylamidates with ß-alanine. BAAT-S immunoprecipitated when incubated with peroxisomal biogenesis factor 5 (Pex5) and rabbit anti-Pex5 antibodies; however, deleting the final 12 AAs prevented coimmunoprecipitation with Pex5, indicating the Pex5 interaction involves more than the -SQL sequence. These results indicate that even small changes in the carboxy terminus of BAAT can have significant effects on activity and substrate specificity.

Dimeric human sulfotransferase 1B1 displays cofactor-dependent subunit communication.

The cytosolic sulfotransferases (SULTs) are dimeric enzymes that catalyze the transformation of hydrophobic drugs and hormones into hydrophilic sulfate esters thereby providing the body with an important pathway for regulating small molecule activity and excretion. While SULT dimerization is highly conserved, the necessity for the interaction has not been established. To perform its function, a SULT must efficiently bind the universal sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), and release the byproduct, 3', 5'-diphosphoadenosine (PAP), following catalysis. We hypothesize this efficient binding and release of PAPS/PAP may be connected to SULT dimerization. To allow for the visualization of dynamic protein interactions critical for addressing this hypothesis and to generate kinetically testable hypotheses, molecular dynamic simulations (MDS) of hSULT1B1 were performed with PAPS and PAP bound to each dimer subunit in various combinations. The results suggest the dimer subunits may possess the capability of communicating with one another in a manner dependent on the presence of the cofactor. PAP or PAPS binding to a single side of the dimer results in decreased backbone flexibility of both the bound and unbound subunits, implying the dimer subunits may not act independently. Further, binding of PAP to one subunit of the dimer and PAPS to the other caused increased flexibility in the subunit bound to the inactive cofactor (PAP). These results suggest SULT dimerization may be important in maintaining cofactor binding/release properties of SULTs and provide hypothetical explanations for SULT half-site reactivity and substrate inhibition, which can be analyzed in vitro.

Activity Suppression Behavior Phenotype in SULT4A1 Frameshift Mutant Zebrafish.

Since its identification in 2000, sulfotransferase (SULT) 4A1 has presented an enigma to the field of cytosolic SULT biology. SULT4A1 is exclusively expressed in neural tissue, is highly conserved, and has been identified in every vertebrate studied to date. Despite this singular level of conservation, no substrate or function for SULT4A1 has been identified. Previous studies demonstrated that SULT4A1 does not bind the obligate sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate, yet SULT4A1 is classified as a SULT superfamily member based on sequence and structural similarities to the other SULTs. In this study, transcription activator-like effector nucleases were used to generate heritable mutations in the SULT4A1 gene of zebrafish. The mutation (SULT4A1(Δ8)) consists of an 8-nucleotide deletion within the second exon of the gene, resulting in a frameshift mutation and premature stop codon after 132 AA. During early adulthood, casual observations were made that mutant zebrafish were exhibiting excessively sedentary behavior during the day. These observations were inconsistent with published reports on activity in zebrafish that are largely diurnal organisms and are highly active during the day. Thus, a decrease in activity during the day represents an abnormal behavior and warranted further systematic analysis. EthoVision video tracking software was used to monitor activity levels in wild-type (WT) and SULT4A1(Δ8/Δ8) fish over 48 hours of a normal light/dark cycle. SULT4A1(Δ8/Δ8) fish were shown to exhibit increased inactivity bout length and frequency as well as a general decrease in daytime activity levels when compared with their WT counterparts.

Structural plasticity in the human cytosolic sulfotransferase dimer and its role in substrate selectivity and catalysis.

The cytosolic sulfotransferases (SULTs) are dimeric enzymes that help maintain homeostasis through the modulation of hormone and drug activity by catalyzing their transformation into hydrophilic sulfate esters and increasing their excretion. Each of the thirteen active human SULT isoforms displays a unique substrate specificity pattern that underlies its individual role in our bodies. These specificities have proven to be complex, in some cases masking the biological role of specific isoforms. The first part of this review offers a short summary of historical underpinnings of human SULTs, primarily centered on the characterization of each isoform's kinetic and structural properties. Recent structural investigations have revealed each SULT has an active site "lid" that undergoes restructuring once the cofactor/sulfonate donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), binds to the enzyme. This structural rearrangement can alter substrate-binding profiles, therefore complicating enzyme/substrate interactions and making substrate/cosubstrate concentrations and binding order important considerations in enzyme functionality. Molecular dynamic simulations have recently been employed to describe this restructuring in an attempt to offer insight to its effects on substrate selectivity. In addition to reviewing new data on SULT molecular dynamics, we will discuss the contribution of PAPS concentrations and SULT dimerization in the regulation of SULT activity within the human body.

The allosteric binding sites of sulfotransferase 1A1.

Human sulfotransferases (SULTs) comprise a small, 13-member enzyme family that regulates the activities of thousands of compounds-endogenous metabolites, drugs, and other xenobiotics. SULTs transfer the sulfuryl-moiety (-SO3) from a nucleotide donor, PAPS (3'-phosphoadenosine 5'-phosphosulfate), to the hydroxyls and primary amines of acceptors. SULT1A1, a progenitor of the family, has evolved to sulfonate compounds that are remarkably structurally diverse. SULT1A1, which is found in many tissues, is the predominant SULT in liver, where it is a major component of phase II metabolism. Early work demonstrated that catechins and nonsteroidal anti-inflammatory drugs inhibit SULT1A1 and suggested that the inhibition was not competitive versus substrates. Here, the mechanism of inhibition of a single, high affinity representative from each class [epigallocatechin gallate (EGCG) and mefenamic acid] is determined using initial-rate and equilibrium-binding studies. The findings reveal that the inhibitors bind at sites separate from those of substrates, and at saturation turnover of the enzyme is reduced to a nonzero value. Further, the EGCG inhibition patterns suggest a molecular explanation for its isozyme specificity. Remarkably, the inhibitors bind at sites that are separate from one another, and binding at one site does not affect affinity at the other. For the first time, it is clear that SULT1A1 is allosterically regulated, and that it contains at least two, functionally distinct allosteric sites, each of which responds to a different class of compounds.

The potent inhibition of human cytosolic sulfotransferase 1A1 by 17α-ethinylestradiol is due to interactions with isoleucine 89 on loop 1.

Drug-drug interactions (DDI) with oral contraceptives containing 17α-ethinylestradiol (EE2) have been well characterized with regard to interactions with phase I drug metaolizing enzymes; however, DDI with EE2 and phase II enzymes have not been as thoroughly addressed. Our laboratory recently reported that in vitro EE2 potently inhibits human cytosolic sulfotransferase (SULT) 1A1 while EE2 was not sulfated until micromolar concentrations. Molecular docking studies demonstrated that Tyr169 and isoleucine 89 (Ile89) may play a role in the inhibitory and/or catalytic positioning of EE2 within the active site of SULT1A1. Therefore, the current study focused on determining the role of Ile89 in the inhibition of SULT1A1 utilizing site-directed mutagenesis. Ile89 was mutated to an alanine and the effect of the mutation was characterized using kinetic and binding assays. SULT1A1-Ile89Ala was found to have a Km for EE2 that was 11-fold greater than wild-type enzyme. A decreased affinity (Kd) of EE2 for SULT1A1-Ile89Ala was apparently responsible for the increase in Km, and also resulted in the loss of the potent inhibition. Molecular modeling was used in an attempt to determine the atomic level changes in binding of EE2 to SULT1A1-Ile89Ala. However, analysis of the effect of the single Ile89 mutation on both the open and closed homology models was not consistent with the docking and kinetic results. Overall, the mechanism of inhibition of EE2 for SULT1A1 is apparently the result of interactions of Ile89 with EE2 holding it in a potent inhibitory conformation, and mutation of the Ile89 significantly decreases the inhibition.

Paradigms of sulfotransferase catalysis: the mechanism of SULT2A1.

Human cytosolic sulfotransferases (SULTs) regulate the activities of thousands of signaling small molecules via transfer of the sulfuryl moiety (-SO3) from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the hydroxyls and primary amines of acceptors. Sulfonation controls the affinities of ligands for their targets, and thereby regulates numerous receptors, which, in turn, regulate complex cellular responses. Despite their biological and medical relevance, basic SULT mechanism issues remain unresolved. To settle these issues, and to create an in-depth model of SULT catalysis, the complete kinetic mechanism of a representative member of the human SULT family, SULT2A1, was determined. The mechanism is composed of eight enzyme forms that interconvert via 22 rate constants, each of which was determined independently. The result is a complete quantitative description of the mechanism that accurately predicts complex enzymatic behavior. This is the first description of a SULT mechanism at this resolution, and it reveals numerous principles of SULT catalysis and resolves previously ambiguous issues. The structures and catalytic behaviors SULTs are highly conserved; hence, the mechanism presented here should prove paradigmatic for the family.

Inhibition of SULT4A1 expression induces up-regulation of phototransduction gene expression in 72-hour postfertilization zebrafish larvae.

Sulfotransferase (SULT) 4A1 is an orphan enzyme that shares distinct structure and sequence similarities with other cytosolic SULTs. SULT4A1 is primarily expressed in neuronal tissue and is also the most conserved SULT, having been identified in every vertebrate investigated to date. Certain haplotypes of the SULT4A1 gene are correlated with higher baseline psychopathology in schizophrenic patients, but no substrate or function for SULT4A1 has yet been identified despite its high level of sequence conservation. In this study, deep RNA sequencing was used to search for alterations in gene expression in 72-hour postfertilization zebrafish larvae following transient SULT4A1 knockdown (KD) utilizing splice blocking morpholino oligonucleotides. This study demonstrates that transient inhibition of SULT4A1 expression in developing zebrafish larvae results in the up-regulation of several genes involved in phototransduction. SULT4A1 KD was verified by immunoblot analysis and quantitative real-time polymerase chain reaction (qPCR). Gene regulation changes identified by deep RNA sequencing were validated by qPCR. This study is the first identification of a cellular process whose regulation appears to be associated with SULT4A1 expression.

Expression of the orphan cytosolic sulfotransferase SULT1C3 in human intestine: characterization of the transcript variant and implications for function.

The cystolic sulfotransferse 1C3 (SULT1C3) gene was identified by computational analysis of the human genome and suggested to contain duplications of its last two exons (7a/b and 8a/b). Although the SULT1C3 isoform containing the more downstream exons 7b and 8b (SULT1C3d) has been expressed in Escherichia coli, crystallized, and characterized for activity, there is currently no evidence that SULT1C3 is expressed in any human tissue. Using reverse-transcription polymerase chain reaction, we detected SULT1C3 mRNA in the colorectal adenocarcinoma cell line (LS180), colon, and small intestine, but the amplified fragment contained the more upstream exons 7a and 8a. 3'-Rapid amplification of cDNA ends (RACE) confirmed that the SULT1C3 transcript expressed in LS180 cells contained exons 7a/8a, whereas 5'-RACE identified a noncoding exon 1. Full-length SULT1C3 transcript containing exons 7a/8a was amplified from LS180 and intestinal RNA, and in vitro transcription-translation of the cloned cDNA indicated that translation primarily began at the first of three in-frame ATG codons. Since SULT1C3 containing exons 7a/8a (SULT1C3a) would differ by 30 amino acids from SULT1C3d containing exons 7b/8b, we considered the functional implications of expressing one or the other isoform by generating structural models based on the reported crystal structure for SULT1C3d. Comparison of the structures indicated that five of the residues forming the substrate-binding pocket differed between the two isoforms, resulting in a change in both electron density and charge distribution along the inner wall of the substrate-binding pocket. These data indicate that SULT1C3 is expressed in human intestine but suggest that the expressed isoform is likely to differ functionally from the isoform that has been previously characterized.

High accuracy in silico sulfotransferase models.

Predicting enzymatic behavior in silico is an integral part of our efforts to understand biology. Hundreds of millions of compounds lie in targeted in silico libraries waiting for their metabolic potential to be discovered. In silico "enzymes" capable of accurately determining whether compounds can inhibit or react is often the missing piece in this endeavor. This problem has now been solved for the cytosolic sulfotransferases (SULTs). SULTs regulate the bioactivities of thousands of compounds--endogenous metabolites, drugs and other xenobiotics--by transferring the sulfuryl moiety (SO3) from 3'-phosphoadenosine 5'-phosphosulfate to the hydroxyls and primary amines of these acceptors. SULT1A1 and 2A1 catalyze the majority of sulfation that occurs during human Phase II metabolism. Here, recent insights into the structure and dynamics of SULT binding and reactivity are incorporated into in silico models of 1A1 and 2A1 that are used to identify substrates and inhibitors in a structurally diverse set of 1,455 high value compounds: the FDA-approved small molecule drugs. The SULT1A1 models predict 76 substrates. Of these, 53 were known substrates. Of the remaining 23, 21 were tested, and all were sulfated. The SULT2A1 models predict 22 substrates, 14 of which are known substrates. Of the remaining 8, 4 were tested, and all are substrates. The models proved to be 100% accurate in identifying substrates and made no false predictions at Kd thresholds of 100 µM. In total, 23 "new" drug substrates were identified, and new linkages to drug inhibitors are predicted. It now appears to be possible to accurately predict Phase II sulfonation in silico.