Molecular Basis of Sulfonylurea Herbicide Inhibition of Acetohydroxyacid Synthase

Abstract

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...

Herbicide-binding sites revealed in the structure of plant acetohydroxyacid synthase

“…With the exception of an additional small two-stranded antiparallel ␤ sheet in the ␣ domain, each domain consists of a six-stranded parallel ␤ sheet surrounded by six to nine ␣ helices. The fold of the C-terminal tail closely resembles that of ScAHAS when in complex with the sulfonylureas (5,6). It is anticipated that this region of AtAHAS is disordered in the absence of herbicide, as it is for the free structure of ScAHAS (4).…”

“…Subsequently, the structure of this enzyme in complex with five sulfonylurea herbicides was determined (5,6). Although ScAHAS is a reasonable model of the plant enzyme, there are some puzzling differences.…”

The carboligation reaction of acetohydroxyacid synthase II: Steady-state intermediate distributions in wild type and mutants by NMR

“…Two different kinds of effects are observed: Mutation of Trp-464 leads to a loss of preference for 2-ketobutyrate (10), whereas mutation of Arg-276, Met-250, or Phe-109 appears to lead to specific decreases in the rates of the product-determining step(s) so that the reaction of the second substrate becomes rate determining (11). We suggested a hypothetical structure for the complex of AHAS II-HEThDP Ϫ with a molecule of 2-ketobutyrate in position to form a new C-C bond (based on the crystal structure of a complex between Saccharomyces cerevisiae AHAS and the herbicide chlorimuron ethyl (20) and on the stereochemistry of the product S-AHAs), which might explain these effects (11). In this hypothetical structure, Trp-464 interacts with the methyl group (C4) of 2-ketobutyrate, whereas Arg-276 is involved in a chargecharge interaction with its carboxylate (Fig.…”

Structure of a Human Carcinogen-converting Enzyme, SULT1A1

“…For example, the type I phosphoribosyltransferase enzymes have a flexible loop that moves to cover the active site during catalysis to protect the transition state from attack by bulk water (41). Similarly, yeast acetohydroxyacid synthase (42,43), yeast pyruvate decarboxylase (44), ribulose-1,5-bisphosphate carboxylase/oxygenase, and triosephosphate isomerase (45) have been shown to undergo disorder-order transitions at the active site.…”