(1/185) Sulfating-activity and stability of cDNA-expressed allozymes of human phenol sulfotransferase, ST1A3*1 ((213)Arg) and ST1A3*2 ((213)His), both of which exist in Japanese as well as Caucasians.
We recently found single amino acid substitutions ((213)Arg/His and (223)Met/Val) in polymorphic human phenol-sulfating phenol sulfotransferase (SULT: cDNAs encoding ST1A3, P PST or HAST1/2) among Caucasians and African-Americans. In a Japanese population (n = 143), allele frequencies of (213)Arg and (213)His were 83.2 and 16. 8%, respectively, but the (223)Val allele was not found. (213)His homozygosity was reportedly associated with both very low (>7-fold) sulfating activities of p-nitrophenol (at 4 microM) and low thermostability in platelets. Sulfating-activity determinations using recombinant (213)Arg- and (213)His-forms (ST1A3*1 and ST1A3*2, respectively) did not, however, reveal appreciable deficiency in [(35)S]3'-phosphoadenosine 5'-phosphosulfate (PAPS)-dependent sulfation of p-nitrophenol (4 microM) by ST1A3*2 (7.5 vs. 10.2 nmol/min/nmol SULT for ST1A3). Kinetic parameters for p-nitrophenol for p-nitrophenol sulfation supported the slight decrease in sulfating activities at 4 microM (K(m), 0.82 vs. 1.75 microM; V(max), 13.2 vs. 13.1 nmol/min/nmol SULT, respectively, for ST1A3*1 and *2). p-Nitrophenyl sulfate-dependent 2-naphthol sulfation by ST1A3*2 was 69% of that by ST1A3*1 (p<0.05). However, ST1A3*2 was remarkably unstable at 45 and 37 degrees C as compared to ST1A3*1. The lower p-nitrophenol sulfating activity of ST1A3*2 may explain the lower platelet p-nitrophenol sulfation in ST1A3*2 homozygotes. Protein instability and ST1A3 gene regulation may be both involved in the polymorphism of p-nitrophenol sulfation in human tissues. (+info)
(2/185) 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. (+info)
(3/185) X-ray crystal structure of human dopamine sulfotransferase, SULT1A3. Molecular modeling and quantitative structure-activity relationship analysis demonstrate a molecular basis for sulfotransferase substrate specificity.
Humans are one of the few species that produce large amounts of catecholamine sulfates, and they have evolved a specific sulfotransferase, SULT1A3 (M-PST), to catalyze the formation of these conjugates. An orthologous protein has yet to be found in other species. To further our understanding of the molecular basis for the unique substrate selectivity of this enzyme, we have solved the crystal structure of human SULT1A3, complexed with 3'-phosphoadenosine 5'-phosphate (PAP), at 2.5 A resolution and carried out quantitative structure-activity relationship (QSAR) analysis with a series of phenols and catechols. SULT1A3 adopts a similar fold to mouse estrogen sulfotransferase, with a central five-stranded beta-sheet surrounded by alpha-helices. SULT1A3 is a dimer in solution but crystallized with a monomer in the asymmetric unit of the cell, although dimer interfaces were formed by interaction across crystallographic 2-fold axes. QSAR analysis revealed that the enzyme is highly selective for catechols, and catecholamines in particular, and that hydrogen bonding groups and lipophilicity (cLogD) strongly influenced K(m). We also investigated further the role of Glu(146) in SULT1A3 using site-directed mutagenesis and showed that it plays a key role not only in defining selectivity for dopamine but also in preventing many phenolic xenobiotics from binding to the enzyme. (+info)
(4/185) Sulfation of "estrogenic" alkylphenols and 17beta-estradiol by human platelet phenol sulfotransferases.
We have investigated the ability of alkylphenols to act as substrates and/or inhibitors of phenol sulfotransferase enzymes in human platelet cytosolic fractions. Our results indicate: (i) straight chain alkylphenols do not interact with the monoamine-sulfating phenol sulfotransferase (SULT1A3); (ii) short chain 4-n-alkylphenols (C < 8) are substrates for the phenol-sulfating enzymes (SULT1A1/2), which exhibit two activity maxima against substrates with alkyl chain lengths of C1-2 and C4-5; (iii) long chain 4-n-substituted alkylphenols (C >/= 8) are poor substrates and act as inhibitors of SULT1A1/2; (iv) human platelets contain two activities, of low and high affinity, capable of sulfating 17beta-estradiol, and 4-n-nonylphenol is a partial mixed inhibitor of the low affinity form of this activity. We conclude that by acting either as substrates or inhibitors of SULT1A1/2, alkylphenols may influence the sulfation, and hence the excretion, of estrogens and other phenol sulfotransferase substrates in humans. (+info)
(5/185) Human phenol sulfotransferases hP-PST and hM-PST activate propane 2-nitronate to a genotoxicant.
The industrial solvent 2-nitropropane (2-NP) is a genotoxic hepatocarcinogen in rats. The genotoxicity of the compound in rats has been attributed to sulfotransferase-mediated formation of DNA-reactive nitrenium ions from the anionic form of 2-NP, propane 2-nitronate (P2N). Whether human sulfotransferases are capable of activating P2N is unknown. In the present study we have addressed this question by investigating the genotoxicity of P2N in various V79-derived cell lines engineered for expression of individual forms of human sulfotransferases, the phenol-sulfating and the monoamine-sulfating phenol sulfotransferases (hP-PST and hM-PST) and the human hydroxysteroid sulfotransferase (hHST). Genotoxicity was assessed by measuring the induction of DNA repair synthesis and by analyzing the formation of DNA modifications. P2N induced repair synthesis in V79-hP-PST and V79-hM-PST cells, whereas induction of repair synthesis in V79-hHST cells was negligible. P2N also resulted in the formation of 8-aminodeoxyguanosine and increased the level of 8-oxodeoxyguanosine in V79-hP-PST cells, but not in the parental V79-MZ cells, which do not show any sulfotransferase activity. Acetone oxime, the tautomeric form of the first reduction product of 2-NP, 2-nitrosopropane, was inactive in all cell lines. The results show that the human phenol sulfotransferases P-PST and M-PST are capable of metabolically activating P2N (P-PST >> M-PST) and that the underlying mechanism is apparently identical to that resulting in the activation of P2N in rat liver, where 2-NP causes carcinomas. These results support the notion that 2-NP should be regarded as a potential human carcinogen. (+info)
(6/185) The ssu locus plays a key role in organosulfur metabolism in Pseudomonas putida S-313.
Pseudomonas putida S-313 can utilize a broad range of aromatic sulfonates as sulfur sources for growth in sulfate-free minimal medium. The sulfonates are cleaved monooxygenolytically to yield the corresponding phenols. miniTn5 mutants of strain S-313 which were no longer able to desulfurize arylsulfonates were isolated and were found to carry transposon insertions in the ssuEADCBF operon, which contained genes for an ATP-binding cassette-type transporter (ssuABC), a two-component reduced flavin mononucleotide-dependent monooxygenase (ssuED) closely related to the Escherichia coli alkanesulfonatase, and a protein related to clostridial molybdopterin-binding proteins (ssuF). These mutants were also deficient in growth with a variety of other organosulfur sources, including aromatic and aliphatic sulfate esters, methionine, and aliphatic sulfonates other than the natural sulfonates taurine and cysteate. This pleiotropic phenotype was complemented by the ssu operon, confirming its key role in organosulfur metabolism in this species. Further complementation analysis revealed that the ssuF gene product was required for growth with all of the tested substrates except methionine and that the oxygenase encoded by ssuD was required for growth with sulfonates or methionine. The flavin reductase SsuE was not required for growth with aliphatic sulfonates or methionine but was needed for growth with arylsulfonates, suggesting that an alternative isozyme exists for the former compounds that is not active in transformation of the latter substrates. Aryl sulfate ester utilization was catalyzed by an arylsulfotransferase, and not by an arylsulfatase as in the related species Pseudomonas aeruginosa. (+info)
(7/185) Mutational analysis of the substrate binding/catalytic domains of human M form and P form phenol sulfotransferases.
Human monoamine (M) form and simple phenol (P) form phenol sulfotransferases (PSTs) are greater than 93% identical in their primary sequences and yet display distinct substrate specificities and other enzymatic properties. Through the generation and characterization of a series of chimeric PSTs, we have previously demonstrated two highly variable regions within their sequences to be responsible for determining their substrate phenotypes (Sakakibara, Y., Takami, Y., Nakayama, T., Suiko, M., and Liu, M.-C. (1998) J. Biol. Chem. 273, 6242-6247). By employing the site-directed mutagenesis technique, the present study aims to identify and quantitatively evaluate the specific amino acid residues critical to the substrate binding and catalysis in these two enzymes. Twelve mutated M-PSTs and seven mutated P-PSTs were generated, expressed, and purified. Enzymatic characterization showed that, of the twelve mutated M-PSTs, mutations at residues Asp-86, Glu-89, and Glu-146 resulted in a dramatic decrease in V(max)/K(m) with dopamine as substrate, being greater than 450 times for the D86A/E89I/E146A mutated M-PST. With p-nitrophenol as substrate, the V(max)/K(m) determined for the D86A/E89I/E146A-mutated M-PST increased more than 25 times and approached that determined for the wild-type P-PST. These results indicated that the concerted action of the three mutated residues (D86A, E89I, and E146A) is sufficient for the conversion of the substrate phenotype of M-PST to that of P-PST. Among the mutated P-PSTs, the I89E- and A146E-mutated P-PSTs displayed considerable deviations in V(max)/K(m) with dopamine or p-nitrophenol as substrate. No corresponding changes, however, were detected with the opposite compound as substrate. These results indicated that, in contrast to M-PST, mutations at Ala-86, Ile-89, and Ala-146 to the corresponding residues in M-PST are not sufficient for rendering the change of P-PST substrate phenotype to that of M-PST. For both M-PSTs and P-PSTs, mutations at Lys-48 or His-108 led to the loss of sulfotransferase activities, indicating their importance in the catalytic mechanism. (+info)
(8/185) In vitro bioactivation of N-hydroxy-2-amino-alpha-carboline.
2-Amino-alpha-carboline (A alpha C) is a mutagenic and carcinogenic heterocyclic amine present in foods cooked at high temperature and in cigarette smoke. The mutagenic activity of A alpha C is dependent upon metabolic activation to N-hydroxy-A alpha C (N-OH-A alpha C); however, the metabolism of N-OH-A alpha C has not been studied. We have synthesized 2-nitro-alpha-carboline and N-OH-A alpha C and have examined in vitro bioactivation of N-OH-A alpha C by human and rodent liver cytosolic sulfotransferase(s) and acetyltransferase(s) and by recombinant human N-acetyltransferases, NAT1 and NAT2. The sulfotransferase-dependent bioactivation of N-OH-A alpha C by human liver cytosol exhibited large inter-individual variation (0.5-75, n = 14) and was significantly higher than bioactivation of N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP). Correlation and inhibition studies suggested that the isoform of sulfotransferase primarily responsible for bioactivation of N-OH-A alpha C in human liver cytosol is SULT1A1. O-Acetyltransferase-dependent bioactivation of N-OH-A alpha C by human liver cytosol also exhibited large inter-individual variation (16-192, n = 18). In contrast to other N-hydroxy heterocyclic amines, which are primarily substrates only for NAT2, both NAT1 and NAT2 catalyzed bioactivation of N-OH-A alpha C. The rate of bioactivation of N-OH-A alpha C by both NAT1 and NAT2 was significantly higher than that for N-OH-PhIP. In rat and mouse liver cytosols, the level of sulfotransferase-dependent bioactivation of N-OH-A alpha C was similar to the level in the high sulfotransferase activity human liver cytosol. The level of O-acetyltransferase-dependent bioactivation of N-OH-A alpha C in rat liver cytosol was also comparable with that in the high acetyltransferase activity human liver cytosol. However, the level of O-acetyltransferase-dependent bioactivation of N-OH-A alpha C in mouse liver cytosol was comparable with that in the low acetyltransferase activity human liver cytosol. In contrast to N-OH-PhIP, bioactivation of N-OH-A alpha C was not inhibited by glutathione S-transferase activity; however, DNA binding of N-acetoxy-A alpha C was inhibited 20% in the presence of GSH. These results suggest that bioactivation of N-OH-A alpha C may be a significant source of DNA damage in human tissues after dietary exposure to AalphaC and that the relative contribution of each pathway to bioactivation or detoxification of N-OH-A alpha C differs significantly from other N-hydroxy heterocyclic or aromatic amines. (+info)