Design and Interpretation of Human Sulfotransferase A1 Assays

Abstract

The human sulfotransferases (SULTs) regulate the activities of hundreds, if not thousands, of small molecule metabolites via transfer of the sulfuryl-moiety (-SO 3 ) from the nucleotide donor, 39-phosphoadenosine 59-phosphosulfate (PAPS) to the hydroxyls and amines of the recipients. Our understanding of the molecular basis of SULT catalysis has expanded considerably in recent years. The basic kinetic mechanism of these enzymes, previously thought to be ordered, has been redefined as random for SULT2A1, a representative member of the superfamily. An active-site cap whose structure and dynamics are highly responsive to nucleotides was discovered and shown to be critical in determining SULT selectivity, a topic of longstanding interest to the field. We now realize that a given SULT can operate in two specificity modes-broad and narrow-depending on the disposition of the cap. More recent work has revealed that the caps of the SULT1A1 are controlled by homotropic allosteric interactions between PAPS molecules bound at the dimer's active sites. These interactions cause the catalytic efficiency of SULT1A1 to vary in a substrate-dependent fashion by as much as two orders of magnitude over a range of PAPS concentrations that spans those found in human tissues. SULT catalysis is further complicated by the fact that these enzymes are frequently inhibited by their substrates. This review provides an overview of the mechanistic features of SULT1A1 that are important for the design and interpretation of SULT1A1 assays.

Tetrahydrobiopterin regulates monoamine neurotransmitter sulfonation

“…The SULT1A3 expression plasmid consists of the SULT1A3 coding region inserted into a triple-tag pGEX-6P expression vector containing an (N-terminal)-His/GST/MBP tag (68,69). The cys-insertion mutants used for regio-specific attachment of maleimide-based labels were constructed as follows: Three single-cys mutants were created by inserting cys into the wild-type SULT 1A3 scaffold, which was not DTNB reactive, at residues E26, E151, and K234.…”

“…SULT expression and purification were performed using protocols that have been described in detail previously (68,69). The proteins (SULTs 1A3, 1A1, 1E1, 2A1, and 2B1b) are >95% pure as judged by Coomassieblue staining of SDS/PAGE.…”

The structure of the catechin-binding site of human sulfotransferase 1A1

“…Protein purification. SULT expression and purification were performed as described previously (21,22). Briefly, transformed E. coli [BL21(DE3)] are grown at 37°C in LB to an OD 600 of ∼0.5.…”

“…Sulfonation often radically alters the interactions of a compound with its target and substantially enhances the target's solubility, transport, and clearance (1, 6, 7). Through these and other mechanisms, SULTs contribute in critical ways to maintaining signaling homeostasis and neutralizing toxins (6,8).Given their wide-ranging roles in regulating cellular processes, it is perhaps expected that SULTs would have evolved means of communicating with their environments-mechanisms that enable them to read and respond to the small-molecule composition of their "milieu." Early screening studies intimated that SULTs might be subject to small-molecule allosteric control (9, 10), and recent work confirms this (11).…”

“…Sulfonation often radically alters the interactions of a compound with its target and substantially enhances the target's solubility, transport, and clearance (1, 6, 7). Through these and other mechanisms, SULTs contribute in critical ways to maintaining signaling homeostasis and neutralizing toxins (6,8).…”