Human phenol sulfotransferases SULT1A2 and SULT1A1

Duggleby

Barnett

et al. 2003

148

Sulfonation catalyzed by sulfotransferase enzymes plays an important role in chemical defense mechanisms against various xenobiotics but also bioactivates carcinogens. A major human sulfotransferase, SULT1A1, metabolizes and/or bioactivates many endogenous compounds and is implicated in a range of cancers because of its ability to modify diverse promutagen and procarcinogen xenobiotics. The crystal structure of human SULT1A1 reported here is the first sulfotransferase structure complexed with a xenobiotic substrate. An unexpected finding is that the enzyme accommodates not one but two molecules of the xenobiotic model substrate p-nitrophenol in the active site. This result is supported by kinetic data for SULT1A1 that show substrate inhibition for this small xenobiotic. The extended active site of SULT1A1 is consistent with binding of diiodothyronine but cannot easily accommodate ␤-estradiol, although both are known substrates. This observation, together with evidence for a disorder-order transition in SULT1A1, suggests that the active site is flexible and can adapt its architecture to accept diverse hydrophobic substrates with varying sizes, shapes and flexibility. Thus the crystal structure of SULT1A1 provides the molecular basis for substrate inhibition and reveals the first clues as to how the enzyme sulfonates a wide variety of lipophilic compounds.Sulfonation is a widely distributed biological reaction catalyzed by members of the supergene family of enzymes called sulfotransferases (SULTs).1 SULTs utilize the sulfonate donor 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS) to catalyze sulfonation, yielding PAP as the desulfonated product of the reaction. These enzymes modify the biological activities of a diverse range of compounds including neurotransmitters, hormones, and drugs. The reaction aids excretion of foreign chemicals but, in some cases, causes bioactivation of carcinogens and mutagens (1-3). SULTs are the focus of intense research in the fields of cancer, drug metabolism, and pharmacogenetics because genetic variation, particularly in isoenzyme SULT1A1, may predispose to lung cancers (4), protect against colorectal cancers (5), and affect the age of onset in breast cancer (6).To date, five distinct mammalian gene families for cytosolic SULTs (SULTs 1-5) have been identified (7), although SULT3 and SULT5 have not been found in humans (8). The most extensive group of the human cytosolic SULTs is the SULT1 family, which includes SULTs 1A1, 1A2, 1A3, 1B1, 1C1, 1C2, and 1E1. Each SULT has distinct substrate preferences but may also exhibit broad and overlapping substrate specificity to span the diversity of chemicals requiring sulfonation. For example, SULT1A3 catalyzes dopamine sulfonation but also accepts p-nitrophenol (pNP) with lower affinity (9, 10). SULT1A1, which shares 93% identity with SULT1A3, also sulfonates dopamine and pNP but with the reverse preference, and in addition it can sulfonate a wide range of hydrophobic molecules (Fig. 1) including xenobiotics and endogenous substrates 3,3Ј-diio...

Section: Discussionsupporting

confidence: 53%

Section: Discussionmentioning

confidence: 99%

Structure of a Human Carcinogen-converting Enzyme, SULT1A1

Duggleby

Barnett

et al. 2003

148

“…Substrate inhibition is characteristic of many SULT enzymes and has been reported previously with human SULTA1 and SULT1A3 with pNP and dopamine, respectively (22)(23)(24). The presence of two substrate molecules in the SULT1A1 structure was postulated to explain the slight cooperativity observed at low concentrations of substrate and the inhibition observed at higher concentrations of pNP.…”

mentioning

confidence: 61%

Active Site Mutations and Substrate Inhibition in Human Sulfotransferase 1A1 and 1A3

Barnett

Tsvetanov

et al. 2004

Human SULT1A1 is primarily responsible for sulfonation of xenobiotics, including the activation of promutagens, and it has been implicated in several forms of cancer. Human SULT1A3 has been shown to be the major sulfotransferase that sulfonates dopamine. These two enzymes shares 93% amino acid sequence identity and have distinct but overlapping substrate preferences. The resolution of the crystal structures of these two enzymes has enabled us to elucidate the mechanisms controlling their substrate preferences and inhibition. The presence of two p-nitrophenol (pNP) molecules in the crystal structure of SULT1A1 was postulated to explain cooperativity at low and inhibition at high substrate concentrations, respectively. In SULT1A1, substrate inhibition occurs with pNP as the substrate but not with dopamine. For SULT1A3, substrate inhibition is found for dopamine but not with pNP. We investigated how substrate inhibition occurs in these two enzymes using molecular modeling, site-directed mutagenesis, and kinetic analysis. The results show that residue Phe-247 of SULT1A1, which interacts with both p-nitrophenol molecules in the active site, is important for substrate inhibition. Mutation of phenylalanine to leucine at this position in SULT1A1 results in substrate inhibition by dopamine. We also propose, based on modeling and kinetic studies, that substrate inhibition by dopamine in SULT1A3 is caused by binding of two dopamine molecules in the active site.

“…A characteristic feature of SULTs is substrate inhibition at high concentrations of their preferred substrates (31,32). We have proposed a kinetic model for pNP substrate inhibition in SULT1A1 based on the crystallographic evidence of two pNP molecules in the active site (11).…”

Section: Discussionmentioning

confidence: 99%

The Structure of Human SULT1A1 Crystallized with Estradiol

Tsvetanov

Duggleby

et al. 2005

107

Human SULT1A1 belongs to the supergene family of sulfotransferases (SULTs) involved in the sulfonation of xeno-and endobiotics. The enzyme is also one of the SULTs responsible for metabolic activation of mutagenic and carcinogenic compounds and therefore is implicated in various cancer forms. Further, it is not well understood how substrate inhibition takes place with rigid fused multiring substrates such as 17␤-estradiol (E2) at high substrate concentrations when subcellular fractions or recombinant enzymes are used. To investigate how estradiol binds to SULT1A1, we co-crystallized SULT1A1 with sulfated estradiol and the cofactor product, PAP (3-phosphoadenosine 5-phosphate). The crystal structure of SULT1A1 that we present here has PAP and one molecule of E2 bound in a nonproductive mode in the active site. The structure reveals how the SULT1A1 binding site undergoes conformational changes to accept fused ring substrates such as steroids. In agreement with previous reports, the enzyme shows partial substrate inhibition at high concentrations of E2. A model to explain these kinetics is developed based on the formation of an enzyme⅐PAP⅐E2 dead-end complex during catalysis. This model provides a very good quantitative description of the rate versus the [E2] curve. This dead-end complex is proposed to be that described by the observed structure, where E2 is bound in a nonproductive mode. Cytosolic sulfotransferases (SULTs)2 are involved in the phase II metabolism of numerous xeno-and endobiotics such as drugs, neurotransmitters, bile acids, and hormones (1, 2). SULTs utilize the cofactor 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS) as the sulfonate (SO 3 Ϫ1 ) donor in such reactions that generally lead to the detoxification of substrates by making them more water-soluble and thereby readily excretable via the kidneys. However, in the case of mutagenic and carcinogenic N-hydroxyarylamines and heterocyclic amines and benzylic alcohols of polycyclic aromatic hydrocarbons, sulfonation is a critical step in their metabolic activation to electrophiles that can bind tissue macromolecules such as DNA (3-6). Further, sulfonation has been shown to activate the antihypertensive and hair growth stimulant minoxidil (7). The overall role that sulfonation plays in disease states such as neurodegeneration and cancer is currently under active investigation (8 -10). SULT1A1 has a broad tissue distribution and has been shown to metabolize a wide range of xenobiotics and play a significant role in the metabolism of estrogens and iodothyronines. In resolving the crystal structure (11) of SULT1A1 we showed that it contains a very hydrophobic L-shaped substrate-binding region, which explains how it can accommodate small planar compounds as well as larger L-shaped aromatics such as iodothyronines. However, in those studies we could not explain how extended fused ring systems, such as 17␤-estradiol (E2), can act as substrates of SULT1A1. In addition, no real information has been provided as to why the metabolism of E2 is inhibited at high...