Human cytosolic sulfotransferase SULT1A1.

Sulfonation is an important conjugation reaction required for a range of biological processes including phase II metabolism, whereby sulfo-conjugation renders a compound more hydrophilic to aid its excretion. The major enzyme responsible for xenobiotic sulfonation is the widely expressed cytosolic sulfotransferase SULT1A1. The SULT1A1 crystal structure has provided insights into this enzyme's substrate specificity and catalytic function, including its role in the sulfonation of endogenous substrates such as oestrogens. Contrary to its metabolic role, SULT1A1 can also bioactivate compounds; it is known to sulfonate pro-carcinogens such as hydroxymethyl polycyclic aromatic hydrocarbons leading to highly reactive intermediates capable of forming DNA adducts, potentially resulting in mutagenesis. Given the role of SULT1A1 in these diverse functions and the discovery of allelic variants with differing catalytic activities, this enzyme has been the focus of numerous polymorphic studies investigating the link between inter-individual SULT1A1 variance and the etiology of a variety of cancers.

Human sulfotransferases and their role in chemical metabolism.

Sulfonation is an important reaction in the metabolism of numerous xenobiotics, drugs, and endogenous compounds. A supergene family of enzymes called sulfotransferases (SULTs) catalyze this reaction. In most cases, the addition of a sulfonate moiety to a compound increases its water solubility and decreases its biological activity. However, many of these enzymes are also capable of bioactivating procarcinogens to reactive electrophiles. In humans three SULT families, SULT1, SULT2, and SULT4, have been identified that contain at least thirteen distinct members. SULTs have a wide tissue distribution and act as a major detoxification enzyme system in adult and the developing human fetus. Nine crystal structures of human cytosolic SULTs have now been determined, and together with site-directed mutagenesis experiments and molecular modeling, we are now beginning to understand the factors that govern distinct but overlapping substrate specificities. These studies have also provided insight into the enzyme kinetics and inhibition characteristics of these enzymes. The regulation of human SULTs remains as one of the least explored areas of research in the field, though there have been some recent advances on the molecular transcription mechanism controlling the individual SULT promoters. Interindividual variation in sulfonation capacity may be important in determining an individual's response to xenobiotics, and recent studies have begun to suggest roles for SULT polymorphism in disease susceptibility. This review aims to provide a summary of our present understanding of the function of human cytosolic sulfotransferases.

The structure of human SULT1A1 crystallized with estradiol. An insight into active site plasticity and substrate inhibition with multi-ring substrates.

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 17beta-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 enzyme x PAP x E2 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.

Active site mutations and substrate inhibition in human sulfotransferase 1A1 and 1A3.

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.

Structure of a human carcinogen-converting enzyme, SULT1A1. Structural and kinetic implications of substrate inhibition.

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 beta-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.