How an enzyme answers multiple-choice questions

The FEBS journal, 2017

Acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6) is the first enzyme in the branched-chain amino acid biosynthesis pathway. Five of the most widely used commercial herbicides (i.e. sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyl-benzoates and sulfonylamino-cabonyl-triazolinones) target this enzyme. Here we have determined the first crystal structure of a plantAHAS in the absence of any inhibitor (2.9 Å resolution) and itshows that the herbicide-binding site adopts a folded state even in the absence of an inhibitor. This is unexpected because the equivalent regions for herbicide bindingin uninhibited Saccharomyces cerevisiae AHAS crystal structures are either disordered,or adopt a different fold when the herbicide is not present. In addition, the structure provides anexplanation as to why some herbicides are more potent inhibitorsofArabidopsis thaliana AHAS compared to AHASs from other species (e.g.Saccharomyces cerevisiae). The elucidation of the native structure of pl...

Plant Physiology and Biochemistry, 2008

Plants and microorganisms synthesize valine, leucine and isoleucine via a common pathway in which the first reaction is catalysed by acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This enzyme is of substantial importance because it is the target of several herbicides, including all members of the popular sulfonylurea and imidazolinone families. However, the emergence of resistant weeds due to mutations that interfere with the inhibition of AHAS is now a worldwide problem. Here we summarize recent ideas on the way in which these herbicides inhibit the enzyme, based on the 3D structure of Arabidopsis thaliana AHAS. This structure also reveals important clues for understanding how various mutations can lead to herbicide resistance.

Pesticide Science, 1990

Several new structurally diverse classes of herbicide, including the sulfonylurea herbicides, act by inhibiting acetolactate synthase, the first common enzyme of branched-chain amino acid biosynthesis. The interaction of acetolactate synthase isozyme 11 (ALSZI) *om Salmonella typhimurium with suEfometuron methyl (SM), a suEfonylurea herbicide, has been used as a paradigm in elucidating the mode of action of these herbicides at the molecular level. A number of diferent studies have collectively suggested that SM binds to ALSII near thiamine pyrophosphate and jlavin adenine dinucleotide (FAD), with its binding site overlapping the second pyruvate (or ketobutyrate) substrate site. Most of S M , however, must be accommodated on ALSII at a site that is not equivalent to substrate or co-factor binding sites. The identity of this herbicide-specific site has recently been suggested by the discovery that poxB, the gene for pyruvate oxidase, shares substantial sequence homology with ilvB, ilvG and ilvI, the genes for the large subunits of ALSI, ALSII, and ALSIII, respectively. Unlike ALSII, pyruvate oxidase uses its FAD for normal redox chemistry, and binds one additional co-factor in vivo, ubiquinone-40. The latter co-factor binds to pyruvate oxidase in a spatially and kinetically equivalent way to SM with ALSII, suggesting that the herbicide binding site of ALSII has a common evolutionary heritage with the ubiquinone site of pyruvate oxidase.

Journal of Molecular Biology, 2002

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) catalyzes the ®rst step in branched-chain amino acid biosynthesis. The enzyme requires thiamin diphosphate and FAD for activity, but the latter is unexpected, because the reaction involves no oxidation or reduction. Due to its presence in plants, AHAS is a target for sulfonylurea and imidazolinone herbicides. Here, the crystal structure to 2.6 A Ê resolution of the catalytic subunit of yeast AHAS is reported. The active site is located at the dimer interface and is near the proposed herbicide-binding site. The conformation of FAD and its position in the active site are de®ned. The structure of AHAS provides a starting point for the rational design of new herbicides.

Bioscience, Biotechnology, and Biochemistry, 2010

The first step in branched-chain amino acid biosynthesis is catalyzed by acetohydroxyacid synthase (EC 2.2.1.6). This reaction involves decarboxylation of pyruvate followed by condensation with either an additional pyruvate molecule or with 2-oxobutyrate. The enzyme requires three cofactors, thiamine diphosphate (ThDP), a divalent ion, and flavin adenine dinucleotide (FAD). Escherichia coli contains three active isoenzymes, and acetohydroxyacid synthase I (AHAS I) large subunit is encoded by the ilvB gene. In this study, the ilvB gene from E. coli K-12 was cloned into expression vector pETDuet-1, and was expressed in E. coli BL21 (DH3). The purified protein was identified on a 12% SDS-PAGE gel as a single band with a mass of 65 kDa. The optimum temperature, buffer, and pH for E. coli K-12 AHAS I were 37 C, potassium phosphate buffer, and 7.5. Km values for E. coli K-12 AHAS I binding to pyruvate, Mg þ2 , ThDP, and FAD were 4.15, 1.26, 0.2 mM, and 0.61 M respectively. Inhibition of purified AHAS I protein was determined with herbicides and new compounds.

Pesticide Biochemistry and Physiology, 1999

Acta Crystallographica Section D Biological Crystallography, 2003

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) catalyses the formation of 2-acetolactate and 2-aceto-2-hydroxybutyrate as the ®rst step in the biosynthesis of the branched-chain amino acids valine, leucine and isoleucine. The enzyme is inhibited by a wide range of substituted sulfonylureas and imidazolinones and many of these compounds are used as commercial herbicides. Here, the crystallization and preliminary X-ray diffraction analysis of the catalytic subunit of Arabidopsis thaliana AHAS in complex with the sulfonylurea herbicide chlorimuron ethyl are reported. This is the ®rst report of the structure of any plant protein in complex with a commercial herbicide. Crystals diffract to 3.0 A Ê resolution, have unitcell parameters a = b = 179.92, c = 185.82 A Ê and belong to space group P6 4 22. Preliminary analysis indicates that there is one monomer in the asymmetric unit and that these are arranged as pairs of dimers in the crystal. The dimers form a very open hexagonal lattice, with a high solvent content of 81%.

Biochemistry, 2004

Acetohydroxy acid synthases (AHAS) are thiamin diphosphate-(ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzymebound FAD proceeds with a net rate constant of k′ ) 0.2 s -1 in the presence of oxygen and approximately 1 s -1 under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)-ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP-and FADdependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer. Martin-Luther-Universität Halle-Wittenberg. § The University of Queensland. | Ben-Gurion University of the Negev. 1 Abbreviations: EcAHAS, acetohydroxy acid synthase from Escherichia coli; YAHAS, acetohydroxy acid synthase from yeast; LpPOX, pyruvate oxidase from Lactobacillus plantarum; EcPOX, pyruvate oxidase from Escherichia coli; ThDP, thiamin diphosphate; HEThDP, 2-(R-hydroxyethyl)thiamin diphosphate; HEThDP -, carbanion/enamine form of HEThDP; AcThDP, 2-acetylthiamin diphosphate; LThDP, 2-lactylthiamin diphosphate; ALThDP, 2-(acetolactyl)thiamin diphosphate; AHBThDP, 2-(acetohydroxybutyryl)thiamin diphosphate; FAD, flavin adenine dinucleotide.

Plant Physiology, 1992

Acetohydroxyacid synthase (AHAS), the first enzyme unique to the biosynthesis of isoleucine, leucine, and valine, is the target enzyme for several classes of herbicides. The AHAS gene from Arabidopsis thaliana, including the chloroplast transit peptide, was cloned into the bacterial expression plasmid pKK233-2. The re- sulting plasmid was used to transform an AHAS-deficient Esche- richia coli strain MF2000. The growth of the MF2000 strain of E. coli was complemented by the functional expression of the Arabidopsis AHAS. The AHAS protein was processed to a molecular mass of 65 kilodaltons that was similar to the mature protein isolated from Arabidopsis seedlings. The AHAS activity extracted from the transformed E. coli cells was inhibited by imidazolinone and sulfonylurea herbicides. AHAS activity extracted from Arabi- dopsis is inhibited by valine and leucine; however, this activity was insensitive to these feedback inhibitors when extracted from the transformed E. coli.

Accounts of Chemical Research, 2001

Acetohydroxy acid isomeroreductase is a key enzyme involved in the biosynthetic pathway of the amino acids isoleucine, valine, and leucine. This enzyme is of great interest in agrochemical research because it is present only in plants and microorganisms, making it a potential target for specific herbicides and fungicides. Moreover, it catalyzes an unusual two-step reaction that is of great fundamental interest. With a view to characterizing both the mechanism of inhibition by potential herbicides and the complex reaction mechanism, various techniques of enzymology, molecular biology, mass spectrometry, X-ray crystallography, and theoretical simulation have been used. The results and conclusions of these studies are described briefly in this paper.