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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The branched-chain amino acids are synthesized by plants, fungi and microorganisms, but not by animals. Therefore, the enzymes of this pathway are potential target sites for the development of antifungal agents, antimicrobials and herbicides. Most research has focused upon the first enzyme in this biosynthetic pathway, acetohydroxyacid synthase (AHAS) largely because it is the target site for many commercial herbicides. In this review we provide a brief overview of the important properties of each enzyme within the pathway and a detailed summary of the most recent AHAS research, against the perspective of work that has been carried out over the past 50 years.

Community-associated methicillin-resistant Staphylococcus aureus of the USA300 lineage is emerging as an important cause of medical device-related infection. However, few factors required for biofilm accumulation by USA300 strains have been identified, and the processes involved are poorly understood. Here, we identify S. aureus proteins required for the USA300 isolate LAC to form biofilm. A mutant with a deletion of the fnbA and fnbB genes did not express the fibronectin-binding proteins FnBPA and FnBPB and lacked the ability to adhere to fibronectin or to form biofilm. Biofilm formation by the mutant LAC∆fnbAfnbB could be restored by expression of FnBPA or FnBPB from a plasmid demonstrating that both of these proteins can mediate biofilm formation when expressed by LAC. Expression of FnBPA and FnBPB increased bacterial aggregation suggesting that fibronectin-binding proteins can promote the accumulation phase of biofilm. Loss of fibronectin-binding proteins reduced the initial adherence of bacteria, indicating that these proteins are also involved in primary attachment. In summary, these findings improve our understanding of biofilm formation by the USA300 strain LAC by demonstrating that the fibronectin-binding proteins are required.

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) is the target for the sulfonylurea herbicides, which act as potent inhibitors of the enzyme. Chlorsulfuron (marketed as Glean) and sulfometuron methyl (marketed as Oust) are two commercially important members of this family of herbicides. Here we report crystal structures of yeast AHAS in complex with chlorsulfuron (at a resolution of 2.19 A), sulfometuron methyl (2.34 A), and two other sulfonylureas, metsulfuron methyl (2.29 A) and tribenuron methyl (2.58 A). The structures observed suggest why these inhibitors have different potencies and provide clues about the differential effects of mutations in the active site tunnel on various inhibitors. In all of the structures, the thiamin diphosphate cofactor is fragmented, possibly as the result of inhibitor binding. In addition to thiamin diphosphate, AHAS requires FAD for activity. Recently, it has been reported that reduction of FAD can occur as a minor side reaction due to reaction with the carbanion/enamine of the hydroxyethyl-ThDP intermediate that is formed midway through the catalytic cycle. Here we report that the isoalloxazine ring has a bent conformation that would account for its ability to accept electrons from the hydroxyethyl intermediate. Most sequence and mutation data suggest that yeast AHAS is a high-quality model for the plant enzyme.