Proposed active site domain in estrogen sulfotransferase as determined by mutational analysis.

Abstract

Point mutations were selectively introduced into a cDNA for guinea pig estrogen sulfotransferase (gpEST); each construct was then expressed in Chinese hamster ovary Kl cells. The molecular site chosen for study is a conserved GXXGXXK sequence that resembles the P-loop-type nucleotide-binding motif for ATP-and GTP-binding proteins and is located near the C terminus of all steroid and phenol (aryl) sulfotransferases for which the primary structures are known. Preliminary experiments demonstrated that the GXXGXXK motif is essential for binding the activated sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The present study was undertaken to ascertain the relative importance of each individual residue of the motif. While the mutation of a single motif residue had little effect on the interaction between gpEST and PAPS as determined by kinetic analysis and photoaffinity labeling, the mutation of any two residues in concert resulted in an approximate 10-fold increase in the Km for PAPS and reduced photoaffinity labeling. The mutation of all three motif residues resulted in an inactive enzyme and complete loss of photoaffinity labeling. Interestingly, several mutants also displayed a striking effect on the Km for the steroid substrate; double mutants, again, demonstrated greater perturbations (8-to 28-fold increase) than did single mutants. Unexpectedly, whereas the mutation of nonmotif residues had a negligible effect on the Km for PAPS, a marked increase in the Km for the estrogen substrate (>30-fold) was noted. On the basis of these findings, it is concluded that the sequence GISGDWKN within the C-terminal domain of gpEST represents a critical component of the active site.The biotransformation of compounds by sulfonation occurs widely, effecting a marked change in the physicochemical properties of the sulfonated products. Sulfonation of drugs and xenobiotics functions primarily to inactivate and clear these generally hydrophobic compounds, although there are examples where the active form of a drug is sulfonated (1). Likewise, the sulfonation of endogenous substances is a fundamental metabolic mechanism that covers an extended range of molecules of diverse size, structure, chemistry, and function. Membrane and secretory proteins are post-translationally modified by sulfonation. Macromolecules such as glycosaminoglycans and proteoglycans, components of cell surface and connective tissue structures, are subjected to modification by the addition of sulfonate groups. Importantly, a multitude of low molecular weight compounds, including neurotransmitters and hormones-e.g., catecholamines, iodothyronines, and steroids, are modified by sulfoconjugation.Inherently, the enzymes that carry out the transfer of a sulfonate group (SO3), termed sulfotransferases, must interact with two substrates, namely, the sulfonate donor and acceptor molecules. In mammals, the universal activated sulfonate donorThe publication costs of this article were defrayed in part by page charge payment. This article must therefor...

Structural and functional characterisation of human sulfotransferases

“…GXXGXXK motif at the C-terminal end of STs [8,9]. Site-directed mutagenesis within this region (GISGDWKN) in the guinea pig estrogen sulfotransferase markedly affected the Km for PAPS and confirmed this part of the amino acid sequence to be a critical component of the active site [9].…”

“…Site-directed mutagenesis within this region (GISGDWKN) in the guinea pig estrogen sulfotransferase markedly affected the Km for PAPS and confirmed this part of the amino acid sequence to be a critical component of the active site [9]. Marsolais and Varin have determined by site-directed mutagenesis of the flavonol 3-sulfotransferase that Arg276, which is in close proximity to the suggested GXXGXXK motif, is crucial for PAPS binding [7].…”

“…However, the change at position 235 in HAST4v compared to HAST4 involves a conservative substitution of an Asn for a Thr. This change is in close proximity to the putative PAPS binding domain [3,8,9] and resides within a region that shows subtle differences in amino acid differences between HAST1, HAST3 and HAST4 proteins. From Table 1 it is apparent that by attaching the last 120 amino acids of HAST4v to HAST4 it was possible to produce a protein that has a Km for PAPS similar to HAST4v.…”

“…The recent cloning of an increasing number of sulfotransferases from a variety of species and analysis of their amino acid sequences has revealed at least four highly conserved regions [2,7]. A number of laboratories have now utilised this data to construct chimeric cDNAs and perform site-directed mutagenesis on these regions, as well as carry out affinity labelling to gain insight into the substrate and PAPS binding sites of guinea pig [8,9], rat [10] and plant [7,11] STs. It has been suggested that the PAPS binding region resides in a highly conserved…”

Human cytosolic sulfotransferase database mining: identification of seven novel genes and pseudogenes

“…1,3 These regions of sequence are important in binding the cosubstrate sulfate donor molecule 3 0 -phosphoadenosine 5 0 -phosphosulfate (PAPS). [4][5][6] In addition, Region IV forms part of the dimerization interface region for these enzymes. 7 SULTs can be classified into families whose members share at least 45% amino-acid sequence identity, and subfamilies that include enzymes that share at least 60% amino-acid sequence identity.…”