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 sulfonylureas and imidazolinones are potent commercial herbicide families. They are among the most popular choices for farmers worldwide, because they are nontoxic to animals and highly selective. These herbicides inhibit branched-chain amino acid biosynthesis in plants by targeting acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This report describes the 3D structure of Arabidopsis thaliana AHAS in complex with five sulfonylureas (to 2.5 Å resolution) and with the imidazolinone, imazaquin (IQ; 2.8 Å). Neither class of molecule has a structure that mimics the substrates for the enzyme, but both inhibit by blocking a channel through which access to the active site is gained. The sulfonylureas approach within 5 Å of the catalytic center, which is the C2 atom of the cofactor thiamin diphosphate, whereas IQ is at least 7 Å from this atom. Ten of the amino acid residues that bind the sulfonylureas also bind IQ. Six additional residues interact only with the sulfonylureas, whereas there are two residues that bind IQ but not the sulfonylureas. Thus, the two classes of inhibitor occupy partially overlapping sites but adopt different modes of binding. The increasing emergence of resistant weeds due to the appearance of mutations that interfere with the inhibition of AHAS is now a worldwide problem. The structures described here provide a rational molecular basis for understanding these mutations, thus allowing more sophisticated AHAS inhibitors to be developed. There is no previously described structure for any plant protein in complex with a commercial herbicide.inhibition ͉ sulfonylurea ͉ x-ray crystallography ͉ imidazolinone ͉ thiamin diphosphate T he sulfonylurea and imidazolinone herbicides are an essential part of the multibillion-dollar weed-control market. There are now Ͼ30 herbicides from these families registered for worldwide use. A major advantage of these compounds is that they are nontoxic to animals, highly selective, and very potent, thereby allowing low application rates. These herbicides act by inhibiting acetohydroxyacid synthase [AHAS; also known as acetolactate synthase; EC 2.2.1.6 (1)], the first common enzyme in the biosynthetic pathway of the branched-chain amino acids. The reaction carried out by this enzyme is the synthesis of either (S)-2-acetolactate from two molecules of pyruvate or (S)-2-aceto-2-hydroxybutyrate from a molecule each of pyruvate and 2-ketobutyrate.AHAS belongs to a superfamily of thiamin diphosphate (ThDP)-dependent enzymes that are capable of catalyzing a variety of reactions, including both the oxidative and nonoxidative decarboxylation of 2-ketoacids. This cofactor is bound by a divalent metal ion such as Mg 2ϩ , which coordinates to the diphosphate group of ThDP and to two highly conserved residues (2) in these proteins. AHAS also binds a molecule of FAD, although this cofactor does not participate in the principal reactions. To date, most AHAS enzymes that have been characterized have both a catalytic subunit (Ϸ65 kDa) and a smaller regulatory subunit, which varies in s...

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Enzymic catalysis proceeds via intermediates formed in the course of substrate conversion. Here, we directly detect key intermediates in thiamin diphosphate (ThDP)-dependent enzymes during catalysis using (1)H NMR spectroscopy. The quantitative analysis of the relative intermediate concentrations allows the determination of the microscopic rate constants of individual catalytic steps. As demonstrated for pyruvate decarboxylase (PDC), this method, in combination with site-directed mutagenesis, enables the assignment of individual side chains to single steps in catalysis. In PDC, two independent proton relay systems and the stereochemical control of the enzymic environment account for proficient catalysis proceeding via intermediates at carbon 2 of the enzyme-bound cofactor. The application of this method to other ThDP-dependent enzymes provides insight into their specific chemical pathways.

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Acetohydroxyacid synthase (AHAS) (acetolactate synthase, EC 4.1.3.18) catalyzes the first step in branchedchain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides. These compounds are potent and selective inhibitors, but their binding site on AHAS has not been elucidated. Here we report the 2.8 Å resolution crystal structure of yeast AHAS in complex with a sulfonylurea herbicide, chlorimuron ethyl. The inhibitor, which has a K i of 3.3 nM, blocks access to the active site and contacts multiple residues where mutation results in herbicide resistance. The structure provides a starting point for the rational design of further herbicidal compounds.Herbicides are widely used for weed control in agriculture and industry and are also used by government agencies and home gardeners. It is estimated that worldwide sales of herbicides exceed $30 billion, with the sulfonylureas (Fig. 1a) and imidazolinones (Fig. 1b) accounting for about $2 billion in annual sales. The sulfonylureas and imidazolinones act by preventing branched-chain amino acid biosynthesis by virtue of their specific and potent inhibition of acetohydroxyacid synthase (AHAS) 1 (acetolactate synthase, EC 4.1.3.18), the first enzyme in this pathway (1, 2).AHAS catalyzes the decarboxylation of pyruvate and its combination with another 2-ketoacid to give an acetohydroxyacid (3, 4). The enzyme requires three cofactors: thiamine diphosphate (ThDP), a divalent metal ion such as Mg 2ϩ , and FAD. The requirement for the first two of these cofactors is well understood from the chemistry of ThDP and the three-dimensional structure of various enzymes including AHAS (5) and its relatives pyruvate oxidase (6), pyruvate decarboxylase (7,8), and benzoylformate decarboxylase (9). In contrast, the role of FAD remains puzzling, despite now knowing the location and conformation of this cofactor in the enzyme (5).The herbicides that inhibit AHAS bear no resemblance to the substrates and are not competitive inhibitors, suggesting that they bind at a site distinct from the active site (1, 10 -13).Previously, we proposed (5) a model for the herbicide-binding site, based on the structure of yeast AHAS and the location of residues where mutation is known to result in herbicide insensitivity. However, this site is large and exposed to solvent, and we suggested that structural changes would occur upon binding of substrates or herbicides. In this paper, we describe the crystal structure of yeast AHAS in complex with chlorimuron ethyl (CE; Fig. 1a), a commonly used sulfonylurea herbicide. Our structure provides the first view of the mode of binding between an herbicidal inhibitor and AHAS and elucidates the location of the herbicide resistance mutations in this enzyme. EXPERIMENTAL PROCEDURESExpression, Purification, Crystallization, and X-ray Data Collection-The catalytic subunit of yeast AHAS was expressed and purified as described previously (14). Crystals of yeast AHAS were grown by hanging drop vapor diffusion in the presence of 1 mM ThDP, 1 mM M...

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