Differential xenoestrogen-sulfating activities of the human cytosolic sulfotransferases: molecular cloning, expression, and purification of human SULT2B1a and SULT2B1b sulfotransferases.

Environmental xenoestrogens have been implicated in human reproductive disorders and an increased incidence of breast cancer. Sulfation, a Phase II detoxification mechanism involving the cytosolic sulfotransferases (STs), may be an important mechanism in vivo for fending off these compounds. In this study, we report on the molecular cloning, expression, and purification of two human cytosolic STs, SULT2B1a and SULT2b1b. The activities of these two enzymes, as well as the other eight known human cytosolic STs previously prepared, toward representative environmental xenoestrogens were examined. Activity data showed that P-form (SULT1A1) PST displayed the highest activity toward these compounds, while SULT1C ST #2 also showed considerable activity, indicating that these enzymes may play a more important role in detoxification of environmental xenoestrogens. SULT1C ST #1, SULT2B1a ST, SULT2B1b ST and NST showed negligible or undetectable activity toward these compounds. The other four enzymes, M-form (SULT1A3) PST, SULT1B2 ST, SULT2A1 ST and SULT1E ST showed intermediate levels of activity toward some of these compounds. Kinetic studies on the sulfation of xenoestrogens by P-form (SULT1A1) PST were performed. The results are interpreted in the context of the endocrine-disrupting nature of these xenoestrogens.

Sulphonation of dehydroepiandrosterone and neurosteroids: molecular cloning, expression, and functional characterization of a novel zebrafish SULT2 cytosolic sulphotransferase.

By searching the zebrafish EST (expressed-sequence tag) database, we have identified two partial cDNA clones encoding the 5' and 3' regions of a putative zebrafish sulphotransferase (ST). Using the reverse transcription-PCR technique, a full-length cDNA encoding this zebrafish ST was successfully cloned. Sequence analysis revealed that this novel zebrafish ST displays 44%, 43% and 40% amino acid identity with mouse SULT2B1, human SULT2B1b and human SULT2A1 ST respectively. This zebrafish ST therefore appears to belong to the SULT2 cytosolic ST gene family. Recombinant zebrafish ST, expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as a 34 kDa protein upon SDS/PAGE. Purified zebrafish ST displayed a strong sulphonating activity toward DHEA (dehydroepiandrosterone), with a optimum pH of 9.5. The enzyme also exhibited activities toward several neurosteroids with differential K(m) and V(max) values. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 degrees C and 43 degrees C. Among ten different divalent metal cations tested, Fe2+ and Cd(2+ exhibited small, but significant, stimulatory effects, whereas Hg2+ and Cu2+ displayed considerably stronger inhibitory effects on the DHEA-sulphonating activity of the enzyme. These results constitute the first study on the molecular cloning, expression, and characterization of a zebrafish cytosolic SULT2 ST.

Highly conserved mouse and human brain sulfotransferases: molecular cloning, expression, and functional characterization.

By employing reverse transcription-polymerase chain reaction (RT-PCR) in conjunction with 5'-rapid amplification of cDNA ends technique, we have cloned a novel mouse sulfotransferase cDNA. Database search led to the identification of a human gene encoding the homologue of this newly discovered mouse sulfotransferase. RT-PCR technique was employed to clone the cDNA encoding the human enzyme. Sequence analysis revealed that the novel mouse and human sulfotransferases display nearly 98% identity in their amino acid sequences. Their amino acid sequence identity to other known cytosolic sulfotransferases, however, was found to be below 36%. These two highly conserved sulfotransferases therefore appear to belong to a family different from the two major mammalian cytosolic sulfotransferase gene families. Northern blot analysis revealed the neuronal tissue-specific expression of these two novel sulfotransferases. Recombinant mouse and human brain sulfotransferases, expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as 33 kD proteins upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified mouse and human brain sulfotransferases displayed enzymatic activities toward endogenous and xenobiotic compounds, including L-triiodothyronine, thyroxine, estrone, p-nitrophenol, 2-naphthylamine, and 2-naphthol. Using mouse brain filtrate as substrate, both brain sulfotransferases were shown to catalyze specifically the sulfation of only a few compounds.

Differential roles of human monoamine (M)-form and simple phenol (P)-form phenol sulfotransferases in drug metabolism.

Cytosolic sulfotransferases (STs) are traditionally known as Phase II drug-metabolizing or detoxifying enzymes that facilitate the removal of drugs and other xenobiotic compounds. In this study, we carried out a systematic investigation on the sulfation of drug compounds by two major human phenol STs (PSTs), the monoamine (M)-form and simple phenol (P)-form PSTs. Activity data obtained showed the differential substrate specificity of the two enzymes for the thirteen drug compounds tested. Kinetic studies revealed that the M-form PST displayed stereoselectivity for the chiral drug, isoproterenol. The effects of divalent metal cations on the activity of the M-form and P-form PSTs toward representative drug compounds were quantitatively evaluated. Results obtained indicated that the drug-sulfating activities of the two human PSTs were partially or completely inhibited or stimulated by the ten divalent metal cations tested at a 5 mM concentration. The two enzymes appeared to be less sensitive to the effects of physiologically more abundant metal cations such as Mg(2+) and Ca(2+), but more sensitive to the detrimental effects of other metal cations that may enter the body as environmental contaminants.

Regulatory effects of divalent metal cations on human cytosolic sulfotransferases.

Cytosolic sulfotransferases (STs), traditionally viewed as Phase II drug-metabolizing or detoxifying enzymes, are increasingly being implicated in the metabolism of endogenous biologically-active molecules. Except for studies on changes in their levels of expression and activity in the early stage of development in mammals, very little is known about how these enzymes are regulated. In this study, the regulatory effects of divalent metal cations on the activity of human cytosolic STs were quantitatively evaluated. Results obtained indicate that all nine human cytosolic STs examined are partially or completely inhibited/stimulated by the ten divalent metal cations tested at 10 mM concentration. Compared with the other metal cations, the inhibitory or stimulatory effect of Mg2+ and Ca2+ on the activities of the human cytosolic STs appeared to be relatively smaller. Concentration-dependent effects of the divalent metal cations were further examined. The IC50 or EC50 values determined for different divalent metal cations were mostly above their normal physiological concentration ranges. In a few cases, however, IC50 values close to the physiological concentrations of certain divalent metal cations were observed. Using the monoamine (M)-form phenol ST (PST) as a model, it was demonstrated that the K(m) for dopamine changed only slightly with increasing concentrations of Cd2+, whereas the V(max) was dramatically decreased.

Structure-function relationships in the stereospecific and manganese-dependent 3,4-dihydroxyphenylalanine/tyrosine-sulfating activity of human monoamine-form phenol sulfotransferase, SULT1A3.

The human monoamine-form phenol sulfotransferase (PST), SULT1A3, has a unique 3,4-dihydroxyphenylalanine (Dopa)/tyrosine-sulfating activity that is stereospecific for their d-form enantiomers and can be stimulated dramatically by Mn(2+). This activity is not present in the simple phenol-form PST, SULT1A1, which is otherwise >93% identical to SULT1A3 in amino acid sequence. The majority of the differences between these two proteins reside in two variable regions of their sequences. Through the characterization of chimeric PSTs where these two regions were exchanged between them, it was demonstrated that variable Region II of SULT1A3 is required for the stereospecificity of its Dopa/tyrosine-sulfating activity, whereas variable Region I of SULT1A3 is required for the stimulation by Mn(2+) of this activity. Further studies using point-mutated SULT1A3s mutated at amino acid residues in these two regions and deletional mutants missing residues 84-86 and 84-90 implicate residue Glu-146 (in variable Region II of SULT1A3), as well as the presence of residues 84-90 of variable Region I, in the stereospecificity in the absence of Mn(2+). Residue Asp-86 (in variable Region I of SULT1A3), on the other hand, is critical in the Mn(2+) stimulation of the Dopa/tyrosine-sulfating activity of SULT1A3. A model is proposed, with reference to the reported x-ray crystal structure of SULT1A3, to explain how the normal role of SULT1A3 in dopamine regulation may be subverted in the presence of Mn(2+). These studies could be relevant in understanding the stereoselective action of SULT1A3 on chiral drugs.

Manganese stimulation and stereospecificity of the Dopa (3,4-dihydroxyphenylalanine)/tyrosine-sulfating activity of human monoamine-form phenol sulfotransferase. Kinetic studies of the mechanism using wild-type and mutant enzymes.

Kinetic studies were performed to dissect the mechanism underlying the remarkable Mn(2+) stimulation of the Dopa/tyrosine-sulfating activity of the human monoamine (M)-form phenol sulfotransferase (PST). The activities and the stimulation by Mn(2+) are highly stereospecific for the d-form enantiomers of tyrosine and Dopa. Analysis of the kinetic results strongly suggests that tyrosine-Mn(2+) and tyrosine-Mn(2+)-tyrosine complexes are obligatory substrates, whereas Dopa-Mn(2+) complexes may be better substrates than Dopa alone. This activation of the Dopa/tyrosine-sulfating activity of M-form PST by Mn(2+) via complex formation between Mn(2+) and the substrate is the first reported case of a regulatory mechanism in this important class of enzymes. Our previous studies using point-mutated M-form PSTs established that the Mn(2+) (in the substrate-Mn(2+) complex) exerts its stimulatory effect by binding predominantly to the Asp-86 residue at the active site. We present here further studies using dopamine as substrate to bolster this conclusion. The possible physiological implications of this rather unusual specificity for the d-amino acid and its derivatives and the stimulation by Mn(2+) are discussed in the context of protective and detoxification mechanisms that may operate in neurodegenerative processes in the brain. The Mn(2+) stimulation of the activity of M-form PST toward d-enantiomers of Dopa/tyrosine may have implications for other substrates (including chiral drugs) and for the other cytosolic sulfotransferases that are involved in the regulation of endogenous metabolites as well as in detoxification.

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.