Localization and Functional Analysis of the Substrate Specificity/Catalytic Domains of Human M-form and P-form Phenol Sulfotransferases

Abstract

Human monoamine (M)-form and simple phenol (P)-form phenol sulfotransferases (PSTs), which are greater than 93% identical in their primary sequences, were used as models for investigating the structural determinants responsible for their distinct substrate specificity and other enzymatic properties. A series of chimeric PSTs were constructed by reciprocal exchanges of DNA segments between cDNAs encoding M-form and P-form PSTs. Functional characterization of the recombinant wild-type M-form, P-form, and chimeric PSTs expressed in Escherichia coli and purified to homogeneity revealed that internal domain-spanning amino acid residues 84 -148 contain the structural determinants for the substrate specificity of either M-form or P-form PST. Data on the kinetic constants (K m , V max , and V max /K m ) further showed the differential roles of the two highly variable regions (Region I spanning amino acid residues 84 -89 and Region II spanning amino acid residues 143-148) in substrate binding, catalysis, and sensitivity to the inhibition by 2,6-dichloro-4-nitrophenol. In contrast to the differential sulfotransferase activities of M-form and P-form PSTs toward dopamine and p-nitrophenol, the Dopa/tyrosine sulfotransferase activities were found to be restricted to M-form, but not P-form, PST. Furthermore, the variable Region II of M-form PST appeared to play a predominant role in determining the Dopa/tyrosine sulfotransferase activities of chimeric PSTs. Kinetic studies indicated the role of manganese ions in dramatically enhancing the binding of D-p-tyrosine to wild-type M-form PST. Taken together, these results pinpoint unequivocally the sequence encompassing amino acid residues 84 -148 to be the substrate specificity/catalytic domain of both M-form and P-form PSTs and indicate the importance of the variable Regions I and II in determining their distinct enzymatic properties.Sulfation is a major pathway for the biotransformation/excretion of drugs and xenobiotics as well as endogenous compounds such as catecholamines, cholesterol, steroid and thyroid hormones, and bile acids (1-3). In mammalian cells, the cytosolic sulfotransferases constitute a group of enzymes that catalyze the transfer of a sulfonate group from the active sulfate, PAPS, 1 to a substrate compound containing either a hydroxyl or an amino group (4). Two essential components of their catalytic actions therefore are the PAPS binding activity (which is common among various sulfotransferases) and the substrate binding activity (which is unique for individual sulfotransferases). Through sequence comparison, two highly conserved regions (YPKSGTXW close to the N terminus and RKGXXGD-WKNXFT near the C terminus) among different cytosolic sulfotransferases had been identified (3). Of these two regions, the latter was shown to be similar to a motif, designated the P-loop, found in the sequences of many ATP-and GTP-binding proteins (5). It was further pointed out (6) that sequences homologous to the C-terminal conserved region are present in PAPSsynthesizing enzymes...

Cloning, Expression, and Characterization of Cytosolic Sulfotransferase Isozymes fromDrosophila melanogaster

“…In comparison with human SULTs, the K m and k cat values of dmSTs are relatively high. 18) The k cat values of the dmSTs are also significantly higher than that of ceST1. 8) These data suggest that the dmSTs effectively metabolize high concentration phenolic xenobiotics and act as a powerful defense system.…”

Structure-Function Relationships in the Stereospecific and Manganese-dependent 3,4-Dihydroxyphenylalanine/ Tyrosine-sulfating Activity of Human Monoamine-form Phenol Sulfotransferase, SULT1A3

“…Preparation of Purified Wild-type and Chimeric SULT1A3/SULT1A1 and Point-mutated SULT1A3s-Wild-type and chimeric SULT1A3 and SULT1A1, cloned/generated and expressed using the pGEX-2TK glutathione S-transferase gene fusion system, were purified using glutathione-Sepharose in conjunction with thrombin cleavage to separate the fusion protein, based on the procedure established previously (7). Pointmutated SULT1A3s were prepared using the QuikChange site-directed mutagenesis kit, expressed using the pGEX-2TK glutathione S-transferase gene fusion system and purified with glutathione-Sepharose followed by thrombin cleavage to separate the fusion protein, as described previously (8).…”

“…1B). Characterization of these chimeras indicated that both Regions I and II were indeed critical for the specificity of SULT1A3 for dopamine and of SULT1A1 for p-nitrophenol (7). To extend the study further, we and others (8,10,11) had, by site-directed mutagenesis, exchanged amino acid residues in these two regions between SULT1A3 and SULT1A1.…”

“…Fig. 1A) and yet vary widely in their substrate specificity and other properties (7)(8)(9). They have thus served as an ideal model system to study structure/function relationships in these proteins.…”

“…1A). Based on the hypothesis that the differences in these two variable regions may account for the distinct properties of SULT1A3 and SULT1A1, we had prepared chimeric proteins (7), where these two regions were reciprocally exchanged (cf. Fig.…”