Expression, Purification, Characterization, and Reconstitution of the Large and Small Subunits of Yeast Acetohydroxyacid Synthase

Pang, Siew Siew; Duggleby, Ronald G.

doi:10.1021/bi983013m

Cited by 97 publications

(125 citation statements)

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“…Q 0 has been shown to inhibit ALS1-3 and here we show that it also inhibits MtAHAS and Arabidopsis thaliana AHAS (AtAHAS) (Fig. 8, A and B), through a mechanism apparently similar to the one involved in inhibition by Q 1 .…”

Section: Resultssupporting

confidence: 60%

The Role of a FAD Cofactor in the Regulation of Acetohydroxyacid Synthase by Redox Signaling Molecules

Lonhienne

García

Guddat

2017

Journal of Biological Chemistry

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Edited by F. Peter GuengerichAcetohydroxyacid synthase (AHAS) catalyzes the first step of branched-chain amino acid (BCAA) biosynthesis, a pathway essential to the lifecycle of plants and microorganisms. This enzyme is of high interest because its inhibition is at the base of the exceptional potency of herbicides and potentially a target for the discovery of new antimicrobial drugs. The enzyme has conserved attributes from its predicted ancestor, pyruvate oxidase, such as a ubiquinone-binding site and the requirement for FAD as cofactor. Here, we show that these requirements are linked to the regulation of AHAS, in relationship to its anabolic function. Using various soluble quinone derivatives (e.g. ubiquinones), we reveal a new path of down-regulation of AHAS activity involving inhibition by oxidized redox-signaling molecules. The inhibition process relies on two factors specific to AHAS: (i) the requirement of a reduced FAD cofactor for the enzyme to be active and (ii) a characteristic slow rate of FAD reduction by the pyruvate oxidase side reaction of the enzyme. The mechanism of inhibition involves the oxidation of the FAD cofactor, leading to a time-dependent inhibition of AHAS correlated with the slow process of FAD re-reduction. The existence and conservation of such a complex mechanism suggests that the redox level of the environment regulates the BCAA biosynthesis pathway. This mode of regulation appears to be the foundation of the inhibitory activity of many of the commercial herbicides that target AHAS. Acetohydroxyacid synthase (AHAS)3 (E.C. 2.2.1.6) catalyzes the first step in de novo branched-chain amino acid (BCAA) biosynthesis, an anabolic pathway present in plants, fungi, and bacteria. AHAS catalyzes the condensation of pyruvate with another molecule of pyruvate, or with 2-ketobutyrate, to produce acetolactate or 2-acetohydroxybutyrate, respectively. In relationship to its essential role in anabolism, AHAS activity is highly regulated by cellular processes. For example, the activity of yeast AHAS (ScAHAS, catalytic subunit ilv2) is stimulated by a regulatory subunit (ilv6). The interaction between these two subunits confers sensitivity to feedback inhibition by valine, an inhibition that is partially reversed by ATP (1, 2) (Fig. 1).Of particular interest, soluble ubiquinones Q 0 (Q 0 ) and Q 1 (Q 1 ) inhibit enteric bacteria AHASs (ALS1-3) and this activity has been attributed to the presence of a quinone binding site derived from the evolution of AHAS from a pyruvate oxidase ancestor (3). The unexplained conservation of this "vestigial" quinone binding site in all AHAS suggests the possibility of its involvement in a, yet undiscovered, physiological role (3). Schloss (4) has also shown that Q 0 is able to oxidize the enzymebound FAD, a cofactor derived from a POX ancestor but without an assigned role in AHAS activity. Here, we demonstrate that (i) the reduction of the FAD cofactor is imperative to activate AHAS, and (ii) the inhibition of AHAS by uquinones is related to the oxidation of FAD. W...

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Section: Resultssupporting

confidence: 60%

The Role of a FAD Cofactor in the Regulation of Acetohydroxyacid Synthase by Redox Signaling Molecules

Lonhienne

García

Guddat

2017

Journal of Biological Chemistry

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“…Expression, 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 MgCl 2 , 1 mM FAD, 1 mM CE, 5 mM dithiothreitol, 0.2 M potassium phosphate, pH 7.0, 0.1 M Tris-HCl, pH 7.0, 0.2 M Li 2 SO 4 , and 0.9 M sodium potassium tartrate.…”

Section: Methodsmentioning

confidence: 99%

Molecular Basis of Sulfonylurea Herbicide Inhibition of Acetohydroxyacid Synthase

Pang

Guddat

Duggleby

2003

Journal of Biological Chemistry

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162

169

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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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“…In the absence of the SSU subunit, the CSU dimer is unstable (Vyazmensky et al 1996), suggesting that in the holoenzyme the AHAS dimer is stabilized by a SSU dimer. The large catalytic subunits also show very weak activity without their associated regulatory subunits (Weinstock et al 1992;Pang and Duggleby 1999). The E. coli AHAS enzyme can be reconstituted with SSU subunits from other organisms (Porat et al 2004), and the reconstituted enzymes form stable heterotetramers.…”

mentioning

confidence: 99%

Crystal structures of TM0549 and NE1324—two orthologs of E. coli AHAS isozyme III small regulatory subunit

et al. 2007

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Crystal structures of two orthologs of the regulatory subunit of acetohydroxyacid synthase III (AHAS, EC 2.2.1.6) from Thermotoga maritima (TM0549) and Nitrosomonas europea (NE1324) were determined by single-wavelength anomalous diffraction methods with the use of selenomethionine derivatives at 2.3 Å and 2.5 Å , respectively. TM0549 and NE1324 share the same fold, and in both proteins the polypeptide chain contains two separate domains of a similar size. Each protein contains a C-terminal domain with ferredoxin-type fold and an N-terminal ACT domain, of which the latter is characteristic for several proteins involved in amino acid metabolism. The ferredoxin domain is stabilized by a calcium ion in the crystal structure of NE1324 and by a Mg(H 2 O) 6 2+ ion in TM0549. Both TM0549 and NE1324 form dimeric assemblies in the crystal lattice.Keywords: acetohydroxyacid synthase; actolactate synthase; regulatory subunit; ACT domain; AHAS; protein refolding Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) catalyzes two physiologically significant reactions in the synthesis of isoleucine, valine, and leucine. In the first reaction, which is an initial step in the synthesis of isoleucine, 2-aceto-2-hydroxy butyrate is produced by condensation of 2-ketobutyrate with pyruvate. In the second reaction, 2-acetolactate is synthesized from two pyruvate molecules, and this provides substrates for synthesis of valine and leucine (Umbarger 1978(Umbarger , 1987Chipman et al. 1998). This pathway of branched-chain amino acid biosynthesis is characteristic for bacteria, fungi, algae, and higher plants, but not for animals. Accordingly, AHAS inhibitors have been developed as herbicides which have found broad application (Short and Colborn 1999). The inhibitors of branched-chain amino acids synthesis are also being tested as potential antituberculosis agents (Grandoni et al. 1998;Zohar et al. 2003;Choi et al. 2005).Three different FAD-dependent AHAS isozymes (I, II, and III) are found in enterobacteria (e.g., Escherichia ), but most other organisms from the bacteria encode only a single AHAS enzyme, similar to isozyme III from E. coli. The acetohydroxyacid synthases are multimeric proteins, and most frequently their biological unit is composed of two catalytic subunits (CSU) and two small regulatory subunits (SSU). In the absence of the SSU subunit, the CSU dimer is unstable (Vyazmensky et al. 1996), suggesting that in the holoenzyme the AHAS dimer is stabilized by a SSU dimer. The large catalytic subunits also show very weak activity without their associated regulatory subunits (Weinstock et al. 1992;Pang and Duggleby 1999). The E. coli AHAS enzyme can be reconstituted with SSU subunits from other organisms (Porat et al. 2004), and the reconstituted enzymes form stable heterotetramers. The properly associated holoenzymes show full catalytic activity, and are also susceptible to valine inhibition.Previous mutagenesis studies (Mendel et al. 2001;Kaplun et al. 2006) revealed that the valine binding sites are located at the dimerizatio...

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Expression, Purification, Characterization, and Reconstitution of the Large and Small Subunits of Yeast Acetohydroxyacid Synthase

Cited by 97 publications

References 76 publications

The Role of a FAD Cofactor in the Regulation of Acetohydroxyacid Synthase by Redox Signaling Molecules

The Role of a FAD Cofactor in the Regulation of Acetohydroxyacid Synthase by Redox Signaling Molecules

Molecular Basis of Sulfonylurea Herbicide Inhibition of Acetohydroxyacid Synthase

Crystal structures of TM0549 and NE1324—two orthologs of E. coli AHAS isozyme III small regulatory subunit

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