An activity-based fluorescent sensor for the detection of the phenol sulfotransferase SULT1A1 in living cells.
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ABSTRACT:
SUBMITTER: Baglia RA
PROVIDER: S-EPMC8190907 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
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Project description:Sulfotransferase isoform 1A1 (SULT1A1) is the most highly expressed hepatic sulfotransferase and is involved in the biotransformation of a wide variety of endo- and xenobiotics. A common single nucleotide polymorphism (SNP) in the coding region of SULT1A1, several proximal promoter SNPs, and copy number variation (CNV) are associated with altered enzymatic activity, but these variants do not fully account for the observed variation of SULT1A1 activity in human populations. In order to identify additional SNPs modulating SULT1A1 activity, we examined the 3'-untranslated region (UTR) of SULT1A1 in 97 liver samples. Direct sequencing revealed that two SNPs in the 3'-UTR (902A > G [rs6839] and 973C > T [rs1042157]) and one SNP in the 3'-flanking region (1307G > A [rs4788068]) were common. These SNPs are in absolute linkage disequilibrium with each other and in tight linkage with SULT1A1 1/2 (linkage coefficient D' 0.83) and are significantly associated with SULT1A1 messenger RNA (p = 0.001, 0.029, 0.021) and enzymatic activity (p = 0.022, 0.012, 0.027). We then examined the collective effects of 3'-UTR SNPs, SULT1A1 1/2, and CNV on SULT1A1 activity in 498 Caucasian and 127 African-American subjects by haplotype analysis. This analysis revealed that SULT1A1 1/2 does not contribute to the variation in SULT1A1 enzymatic activity when the 3'-UTR SNPs are included in the statistical model. Two major haplotypes (ACG and GTA) were significantly correlated with SULT1A1 activity, and when stratified by copy number, the SULT1A1 3'-UTR SNPs remain significantly associated with SULT1A1 enzymatic activity in Caucasians, but not in African-Americans. Subsequent functional characterization revealed that a microRNA, miR-631, regulates SULT1A1 expression in a genotype-specific manner.
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