Structure and localization of the human SULT1B1 gene: neighborhood to SULT1E1 and a SULT1D pseudogene.

The soluble sulfotransferases are involved in the elimination of xenobiotics, the activation of procarcinogens, and the regulation of hormones. They comprise a gene superfamily (SULT). The structure and chromosomal location of nine human SULT genes are known. We have characterized a further gene, SULT1B1. Its structure is similar to that of other SULT1 genes. However, the total length of its eight exons and the introns (33.6 kb) is larger than that of other human SULT1 genes (4 to 21 kb). The SULT1B1 gene sequence is part of a sequence entry in the unfinished High-Throughput Genomic Sequences (HTGS) division of GenBank. However, the order and orientation of the SULT1B1 exons are not correct in this entry. SULT1B1 is located on chromosome 4q13.1, nearly 100 kb downstream of SULT1E1 on the same strand. The intervening sequence contains a SULT-like structure showing substantial homology to the mouse SULT1D1 cDNA recently described. However, in humans this structure represents a pseudogene (SULT1D1P) because of mutated splice donors/acceptors and in-frame stop codons in the sequence corresponding to exon II. This SULT gene cluster is located on the minus strand of chromosome 4 with SULT1B1 being closest to the centromer.     

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.     

Enzymatic properties, tissue-specific expression, and lysosomal location of two highly homologous rat SULT1C2 sulfotransferases

We have isolated two highly homologous but distinct rat sulfotransferase cDNAs termed ratSULT1C2 and ratSULT1C2A encoding polypeptides of 297 amino acids each. The amino acid sequence of ratSULT1C2 is 84% identical to the human SULT1C2 and 81% identical to a rabbit SULT1C2 sulfotransferase. ratSULT1C2 and ratSULT1C2A are 92% identical but differ in 22 amino acids. The majority of these amino acid substitutions in ratSULT1C2A is not found in the human and rabbit SULT1C2, which identifies ratSULT1C2 as the orthologue of these sulfotransferases, whereas SULT1C2A is a closely related but distinct enzyme. ratSULT1C2 and 2A sulfotransferases do not sulfonate steroids, dopamine, acetaminophen, or alpha-naphthol, but only p-nitrophenol. Prokaryotically expressed ratSULT1C2A is less active than ratSULT1C2. ratSULT1C2/2A mRNAs are abundant in kidney and less abundant in stomach and liver. The enzymes are expressed as 34-kDa polypeptides in rat kidney, liver, and stomach. In addition, a 28-kDa cross-reacting polypeptide is found in kidney only. Immunohistochemistry revealed expression of ratSULT1C2/2A in the epithelial cells of the proximal tubules of the kidney, bile duct epithelia, hepatocytes, and the epithelium of the gastric mucosal glands. Although the cDNA predicted amino acid sequence identifies both sulfotransferases as cytosolic enzymes, in tissue sections, in the kidney cell line NRK 52, and in transiently transfected BHK cells a considerable fraction of the enzyme was found in a granular perinuclear compartment. Costaining with a lysosomal marker in gastric mucosa tissue sections and cultured cells identifies these structures as lysosomes.     

Localization of a brain sulfotransferase, SULT4A1, in the human and rat brain: an immunohistochemical study.

Cytosolic sulfotransferases are believed to play a role in the neuromodulation of certain neurotransmitters and drugs. To date, four cytosolic sulfotransferases have been shown to be expressed in human brain. Recently, a novel human brain sulfotransferase has been identified and characterized, although its role and localization in the brain are unknown. Here we present the first immunohistochemical (IHC) localization of SULT4A1 in human brain using an affinity-purified polyclonal antibody raised against recombinant human SULT4A1. These results are supported and supplemented by the IHC localization of SULT4A1 in rat brain. In both human and rat brains, strong reactivity was found in several brain regions, including cerebral cortex, cerebellum, pituitary, and brainstem. Specific signal was entirely absent on sections for which preimmune serum from the corresponding animal, processed in the same way as the postimmune serum, was used in the primary screen. The findings from this study may assist in determining the physiological role of this SULT isoform.     

Sulfation of nitrotyrosine: biochemistry and functional implications

Nitration of tyrosine, in both protein-bound form and free amino acid form, can readily occur in cells under oxidative/nitrative stress. In addition to serving as a biomarker of oxidative/nitrative stress, elevated levels of nitrotyrosine have been shown to cause DNA damage or trigger apoptosis. An important issue is whether the human body is equipped with mechanisms to counteract the potentially harmful effects of nitrotyrosine. Sulfate conjugation, as mediated by the cytosolic sulfotransferases (SULTs), is widely used for the biotransformation and disposal of a variety of drugs and other xenobiotics, as well as endogenous thyroid/steroid hormones and catecholamine neurotransmitters. Recent studies have revealed that the sulfation of nitrotyrosine occurs in cells under oxidative/nitrative stress, and have pinpointed the SULT1A3 as the responsible SULT enzyme. In this review, we summarized the available information concerning the biochemistry of nitrotyrosine sulfation and the effects of genetic polymorphisms on the nitrotyrosine sulfating activity of SULT1A3. Functional implications of the sulfation of nitrotyrosine are discussed.     

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.     

Photoaffinity labeling probe for the substrate binding site of human phenol sulfotransferase (SULT1A1): 7-azido-4-methylcoumarin.

A novel fluorescent photoactive probe 7-azido-4-methylcoumarin (AzMC) has been characterized for use in photoaffinity labeling of the substrate binding site of human phenol sulfotransferase (SULT1A1 or P-PST-1). For the photoaffinity labeling experiments, SULT1A1 cDNA was expressed in Escherichia coli as a fusion protein to maltose binding protein (MBP) and purified to apparent homogeneity over an amylose column. The maltose moiety was removed by Factor Xa cleavage. Both MBSULT1A1 and SULT1A1 were efficiently photolabeled with AzMC. This labeling was concentration dependent. In the absence of light, AzMC competitively inhibited the sulfation of 4MU catalyzed by SULT1A1 (Ki = 0.47 +/- 0.05 mM). Moreover, enzyme activity toward 2-naphthol was inactivated in a time- and concentration-dependent manner. SULT1A1 inactivation by AzMC was protected by substrate but was not protected by cosubstrate. These results indicate that photoaffinity labeling with AzMC is highly suitable for the identification of the substrate binding site of SULT1A1. Further studies are aimed at identifying which amino acids modified by AzMC are localized in the binding site.     

Polychlorobiphenylols are selective inhibitors of human phenol sulfotransferase 1A1 with 4-nitrophenol as a substrate

Polychlorobiphenylols (OH-PCBs) were reported as potent inhibitors of estrogen sulfotransferase, thyroid hormone and 3-hydroxybenzo(a)pyrene sulfotransferases. The aim of this study was to examine the effects of selected OH-PCBs on SULT1A1 activity in human liver cytosol, measured with 4microM 4-nitrophenol, a concentration considered to be diagnostic for selectively detecting SULT1A1. All the OH-PCBs studied inhibited the sulfonation of 4-nitrophenol in human liver cytosol. Among the eighteen OH-PCBs studied, 3'-OH-CB3 (4-chlorobiphenyl-3'-ol) was the most potent inhibitor (IC(50): 0.73+/-0.15microM, mean+/-S.D., n=3). The least potent inhibitor studied was 6'-OH-CB35 (3,3',4-trichlorobiphenyl-6'-ol) with IC(50): 49.1+/-10.8microM. The IC(50) values of the other OH-PCBs studied ranged from 0.78 to 3.76microM. Some OH-PCBs with various inhibitory potencies with human liver cytosol were selected for study with recombinant human SULT1A1 and SULT1B1. These OH-PCBs showed more potent inhibition of 4-nitrophenol sulfonation with SULT1A1 than with human liver cytosol. The IC(50) values with human liver cytosol showed a perfect linear correlation with those found with SULT1A1 (r(2)=1), but not with SULT1B1 (r(2)=0.21). The results suggested that in these human samples SULT1A1 was predominantly responsible for the sulfonation of 4-nitrophenol, with very little or no contribution from SULT1B1. The kinetics of inhibition were studied with 4'-OH-CB165, which is similar in structure to OH-PCBs found in human blood. The 4'-OH-CB165 was a mixed noncompetitive-uncompetitive inhibitor (K(i)=1.80+/-0.2microM, K(ies)=0.16+/-0.02microM). Finally, it was demonstrated that the tested OH-PCBs were themselves only slowly sulfonated by human sulfotransferases in the presence of (35)S-PAPS, as measured by the production of (35)S-labeled metabolites. Although this series of 18 OH-PCBs was too small to draw conclusions about structure-potency relationships, this work demonstrated that several OH-PCBs were potent inhibitors of 4-nitrophenol sulfonation but poor substrates in human liver cytosol, and suggested that OH-PCBs may inhibit the sulfation rate of those xenobiotics sulfated by SULT1A1.     

Analysis of the substrate specificity of human sulfotransferases SULT1A1 and SULT1A3: site-directed mutagenesis and kinetic studies.

Sulfonation is an important metabolic process involved in the excretion and in some cases activation of various endogenous compounds and xenobiotics. This reaction is catalyzed by a family of enzymes named sulfotransferases. The cytosolic human sulfotransferases SULT1A1 and SULT1A3 have overlapping yet distinct substrate specificities. SULT1A1 favors simple phenolic substrates such as p-nitrophenol, whereas SULT1A3 prefers monoamine substrates such as dopamine. In this study we have used a variety of phenolic substrates to functionally characterize the role of the amino acid at position 146 in SULT1A1 and SULT1A3. First, the mutation A146E in SULT1A1 yielded a SULT1A3-like protein with respect to the Michaelis constant for simple phenols. The mutation E146A in SULT1A3 resulted in a SULT1A1-like protein with respect to the Michaelis constant for both simple phenols and monoamine compounds. When comparing the specificity of SULT1A3 toward tyramine with that for p-ethylphenol (which differs from tyramine in having no amine group on the carbon side chain), we saw a 200-fold preference for tyramine. The kinetic data obtained with the E146A mutant of SULT1A3 for these two substrates clearly showed that this protein preferred substrates without an amine group attached. Second, changing the glutamic acid at position 146 of SULT1A3 to a glutamine, thereby neutralizing the negative charge at this position, resulted in a 360-fold decrease in the specificity constant for dopamine. The results provide strong evidence that residue 146 is crucial in determining the substrate specificity of both SULT1A1 and SULT1A3 and suggest that there is a direct interaction between glutamic acid 146 in SULT1A3 and monoamine substrates.     

Sulfation of hydroxychlorobiphenyls. Molecular cloning, expression, and functional characterization of zebrafish SULT1 sulfotransferases.

As a first step toward developing a zebrafish model for investigating the role of sulfation in counteracting environmental estrogenic chemicals, we have embarked on the identification and characterization of cytosolic sulfotransferases (STs) in zebrafish. By searching the zebrafish expressed sequence tag database, we have identified two cDNA clones encoding putative cytosolic STs. These two zebrafish ST cDNAs were isolated and subjected to nucleotide sequencing. Sequence data revealed that the two zebrafish STs are highly homologous, being approximately 82% identical in their amino acid sequences. Both of them display approximately 50% amino acid sequence identity to human SULT1A1, rat SULT1A1, and mouse SULT1C1 ST. These two zebrafish STs therefore appear to belong to the SULT1 cytosolic ST gene family. Recombinant zebrafish STs (designated SULT1 STs 1 and 2), expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as approximately 35 kDa proteins on SDS/PAGE. Purified zebrafish SULT1 STs 1 and 2 displayed differential sulfating activities toward a number of endogenous compounds and xenobiotics including hydroxychlorobiphenyls. Kinetic constants of the two enzymes toward two representative hydroxychlorobiphenyls, 3-chloro-4-biphenylol and 3,3',5,5'-tetrachloro-4,4'-biphenyldiol, and 3,3',5-triiodo-l-thyronine were determined. A thermostability experiment revealed the two enzymes to be relatively stable over the range 20-43 degrees C. Among 10 different divalent metal cations tested, Co2+, Zn2+, Cd2+, and Pb2+ exhibited considerable inhibitory effects, while Hg2+ and Cu2+ rendered both enzymes virtually inactive.     

A nucleotide-gated molecular pore selects sulfotransferase substrates

Human SULT2A1 is one of two predominant sulfotransferases in liver and catalyzes transfer of the sulfuryl moiety (-SO(3)) 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 SULT2A1 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 closed 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 nucleotide-driven isomerization controls access to the acceptor-binding pocket and plays an important role in substrate selection by SULT2A1 and possibly other sulfotransferases.     

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.     

Crystal structure of mSULT1D1, a mouse catecholamine sulfotransferase

In mammals, sulfonation as mediated by specific cytosolic sulfotransferases (SULTs) plays an important role in the homeostasis of dopamine and other catecholamines. To gain insight into the structural basis for dopamine recognition/binding, we determined the crystal structure of a mouse dopamine-sulfating SULT, mouse SULT1D1 (mSULT1D1). Data obtained indicated that mSULT1D1 comprises of a single α/β domain with a five-stranded parallel β-sheet. In contrast to the structure of the human SULT1A3 (hSULT1A3)-dopamine complex previously reported, molecular modeling and mutational analysis revealed that a water molecule plays a critical role in the recognition of the amine group of dopamine by mSULT1D1. These results imply differences in substrate binding between dopamine-sulfating SULTs from different species.     

Characterization and tissue distribution of a novel human cytochrome P450-CYP2U1

A novel human cytochrome P450 cDNA designated CYP2U1 was identified using homology searches, and the corresponding gene is located on chromosome 4. The deduced 544 amino acid sequence displays up to 39% identity to other CYP2 family members, with closest resemblance to CYP2R1 and is highly conserved between species. CYP2U1 shows some structural differences compared to other CYP2 family members. The gene has only five exons and the enzyme harbors two insertions in the N-terminal region. Northern blot analysis revealed high mRNA expression in human thymus, with weaker expression in heart and brain, whereas in the rat similar mRNA levels were detected in thymus and brain. Western blot analysis revealed much higher CYP2U1 protein expression in rat brain than in thymus, particularly in limbic structures and in cortex. The physiological and toxicological role of this novel P450 is still unknown, but the selective tissue distribution suggests an important endogenous function.