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Human cytosolic sulfotransferase 1C4 (hSULT1C4) is a dimeric Phase II drug-metabolizing enzyme primarily expressed in the developing fetus. SULTs facilitate the transfer of a hydrophilic sulfonate moiety from 3′-phosphoadenosine-5′-phosphosulfate (PAPS) onto an acceptor substrate altering the substrate’s biological activity and increasing the compound’s water solubility. While several of the hSULTs’ endogenous and xenobiotic substrates have been identified, the physiological function of hSULT1C4 remains unknown. The fetal expression of hSULT1C4 leads to the hypothesis that the function of this enzyme may be to regulate metabolic and hormonal signaling molecules, such as estrogenic compounds, that may be generated or consumed by the mother during fetal development. Human SULT1C4 has previously been shown to sulfonate estrogenic compounds, such as catechol estrogens; therefore, this study focused on the expression and purification of hSULT1C4 in order to further characterize this enzyme’s sulfonation of estrogenic compounds. Molecular modeling of the enzyme’s native properties helped to establish a novel purification protocol for hSULT1C4. The optimal activity assay conditions for hSULT1C4 were determined to be pH 7.4 at 37°C for up to 10 min. Kinetic analysis revealed the enzyme’s reduced affinity for PAPS compared to PAP. Human SULT1C4 sulfonated all the estrogenic compounds tested, including dietary flavonoids and environmental estrogens; however, the enzyme has a higher affinity for sulfonation of flavonoids. These results suggest hSULT1C4 could be metabolizing and regulating hormone signaling pathways during human fetal development.

1. Human cytosolic sulfotransferase 1B1 (SULT1B1) sulfates small phenolic compounds and bioactivates polycyclic aromatic hydrocarbons. To date, no SULT1B1 allelic variants have been well-characterized. 2. While cloning SULT1B1 from human endometrial specimens, an allelic variant resulting in valine instead of leucine at the 145th amino acid position (L145V) was detected. NCBI reported this alteration as the highest frequency SULT1B1 allelic variant. 3. L145V frequency comprised 9% of 37 mixed-population human patients and was specific to African Americans with an allelic frequency of 25%. Structurally, replacement of leucine with valine potentially destabilizes a conserved helix (α8) that forms the "floor" of both the substrate and PAPS binding domains. This destabilization results in altered kinetic properties including a four-fold decrease in affinity for PAP (3', 5'-diphosphoadenosine). Ks for 3'-phosphoadenosine- 5'-phosphosulfate (PAPS) are similar; however, maximal turnover rate of the variant isoform (0.86 pmol/(min*μg)) is slower than wild-type (WT) SULT1B1 (1.26 pmol/(min*μg)). The L145V variant also displays altered kinetics toward small phenolic substrates, including a diminished p-nitrophenol K and increased susceptibility to 1-naphthol substrate inhibition. 4. No significant correlation between genotype and prostate or colorectal cancer was observed in patients; however, the variant isoform could underlie specific pathologies in sub-Saharan African carriers.

Human cytosolic sulfotransferases (hSULTs) are phase II conjugation enzymes that play a critical role in the metabolism of drugs and hormones, the bioactivation of carcinogens, and the detoxification of xenobiotics. SULTs transfer a sulfuryl‐moiety from the obligate donor PAPS (3′‐phosphoadenosine 5′‐phosphosulfate) to a suitable hydroxyl or amine group of the substrate. While some of the SULTs have been studied extensively, others, such as the SULT 1C family, remain poorly characterized. The objective of this study is to determine the role of hSULT1C4 in drug metabolism by identifying novel substrates and defining the substrate binding, selectivity, and activity of the SULT1C4 enzyme using molecular modeling and kinetic analysis. SULT1C4 was bacterially expressed and purified for in vitro kinetic analysis. Steady‐state kinetic parameters for 1‐naphthol were analyzed using four concentrations of 1‐naphthol (0.5, 1, 2.5, and 7.5 uM) and four concentrations of PAPS (0.5, 1.5, 5, 15 uM). The Km of 1‐naphthol was approximately 1 uM, whereas the data for PAPS suggests the existence of two Kms (1 uM and 12 uM). To determine if these two Kms are due to SULT1C4's dimeric protein state, two different dimerization domain mutants, SULT1C4‐K272A and SULT1C4‐V277E, have been generated. Native page gel analysis has shown that these mutations prevent dimerization resulting in monomeric SULT1C4 proteins. Our data suggests that the dimeric state of SULT1C4 influences PAPS’ ability to bind to the enzyme.Funding sources: NIH ES022606 and GM038953