• BMB Reports
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• 제33권1호
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• pp.1-36
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• 2000

Acetohydroxyacid synthase (EC 4.1.3.18) catalyses the first reaction in the pathway for synthesis of the branched-chain amino acids. The enzyme is inhibited by several commercial herbicides and has been subjected to detailed study over the last 20 to 30 years. Here we review the progress that has been made in understanding its structure, regulation, mechanism, and inhibition.

• BMB Reports
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• 제33권3호
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• pp.195-201
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• 2000

A human gene has been reported that may encode the enzyme acetohydroxyacid synthase. Previously this enzyme was thought to be absent from animals although it is present in plants and many microorganisms. In plants, this enzyme is the target of a number of commercial herbicides and the use of these compounds may need to be reassessed if the human enzyme exists and proves to be susceptible to inhibition. Here we report the construction of several plasmid vectors containing the cDNA sequence for this protein, and their expression in Escherichia coli. High levels of expression were observed, but most of the protein proved to be insoluble. The small amounts of soluble protein contained little or no acetohydroxyacid synthase activity. Attempts to refold the insoluble protein were successful insofar as the protein became soluble. However, the refolded protein did not gain any acetohydroxyacid synthase activity. In vivo complementation tests of an E. coli mutant produced no evidence that the protein is active. Incorrect folding, or the lack of another subunit, may explain the data but we favor the interpretation that this gene does not encode an acetohydroxyacid synthase.

• Bulletin of the Korean Chemical Society
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• 제29권9호
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• pp.1823-1826
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• 2008

• Bulletin of the Korean Chemical Society
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• 제27권10호
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• pp.1697-1700
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• 2006

• Bulletin of the Korean Chemical Society
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• 제31권12호
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• pp.3782-3784
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• 2010

• Bulletin of the Korean Chemical Society
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• 제28권7호
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• pp.1109-1113
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• 2007

Acetohydroxyacid synthase (AHAS, EC 2. 2. 1. 6) is the enzyme that catalyses the first step in the common pathway of the biosynthesis of the branched chain amino acids, valine, leucine and isoleucine. For the first time, the AHAS gene from Bacillus anthracis was cloned into the expression vector pET28a(+), and was expressed in the E. coli strain BL21(DE3). The purified enzyme was checked on 12% SDS-PAGE to be a single band with molecular weight of 65 kDa. The optimum pH and temperature for B. anthracis AHAS was at pH 7.5 and 37 oC, respectively. Kinetic parameters of B. anthracis were as follows: Km for pyruvate, K0.5 for ThDP and Mg2+ was 4.8, 0.28 and 1.16 mM respectively. AHAS from B. anthracis showed strong resistance to three classes of herbicides, Londax (a sulfonylurea), Cadre (an imidazolinone), and TP (a triazolopyrimidine). These results indicated that these herbicides could be used in the search for new anti-bacterial drugs.

• Bulletin of the Korean Chemical Society
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• 제26권2호
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• pp.260-264
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• 2005

The nature of the active site of Tobacco acetohydroxyacid synthase (AHAS) in the substrate- and cofactorbinding was studied by kinetics and fluorescence spectroscopy. The substrate saturation curve does not follow Michaelis-Menten kinetics at different temperatures (7, 21 and 37 ${^{\circ}C}$), pH (6.5, 7.5 and 8.5) and buffers (Tris-HCl and MOPS). The concentration of one half of the maximum velocity ($S_{0.5}$) decreased in the following order: pyruvate $\gt$ ThDP $\approx$$Mg^{+2}$ $\gt$ FAD. However, the catalytic efficiency (K$_{cat}/S_{0.5}$) inversely decreased in the following order; FAD $\gt$ $Mg^{+2}$ $\approx$ThDP $\gt$ pyruvate, indicating that the cofactors by in decreasing order; FAD, $Mg^{+2}$, ThDP, affect the catalysis of AHAS. The dissociation constant ($K_d$) of the intrinsic tryptophan fluorescence decreased with the same tendency of the concentration of one half of the maximum velocity ($S_{0.5}$) decreasing order. This data provides evidence that the substrate and cofactor binding natures of the active site, as well as its activation characteristics, resemble those of other ThDP-dependent enzymes.

• Bulletin of the Korean Chemical Society
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• 제33권12호
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• pp.4074-4078
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• 2012

The first step in the common pathway for the biosynthesis of branched chain amino acids is catalyzed by acetohydroxyacid synthase (AHAS). The AHAS is found in plants, fungi and bacteria. With an aim to identify new anti-tuberculosis drugs that inhibit branched chain amino acid biosynthesis, we screened a chemical library against Mycobacterium tuberculosis AHAS. The screening identified four compounds, AVS 2087, AVS 2093, AVS 2236, and AVS 2387 with $IC_{50}$ values of 0.28, 0.21, 3.88, and $0.25{\mu}M$, respectively. Moreover, these four compounds also showed strong inhibition against reconstituted AHAS with $IC_{50}$ values of 0.37, 0.26, 1.0, and $1.18{\mu}M$, respectively. The basic scaffold of the AVS group consists of 1-pyrimidin-2-yl-1H-[1,2,4]-triazole-3-sulfonamide. The most active compound, AVS 2387, showed the lowest total interaction energy -8.75 Kcal/mol and illustrates its binding mode by hydrogen bonding with $H_{\varepsilon}$ of Gln517 with the distance of $2.24{\AA}$.

• Bulletin of the Korean Chemical Society
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• 제27권4호
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• pp.549-555
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• 2006

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6 also referred to as acetolactate synthase) catalyzes the first common step in the metabolic pathway leading to biosynthesis of the branched-chain amino acids in plants and microorganisms. Due to its presence in plants, AHAS is a target for the herbicides (sulfonylurea and imidazolinone), which act as potent inhibitors of the enzyme. Recently, we have shown [J. Kim, D.G. Baek, Y.T. Kim, J.D. Choi, M.Y. Yoon, Biochem. J. (2004) 384, 59-68] that the residues in the “mobile loop” 567-582 on the C-termini are involved in the binding/stabilization of the active dimer and ThDP (thiamin diphosphate) binding. In this study, we have demonstrated the role of the W573 in the mobile loop of the C-termini of tobacco AHAS. The substitution of this W573 residue caused significant perturbations in the activation process and in the binding site of ThDP. Position W573 plays a structurally important role in the binding of FAD, maintaining the enzyme active site in the required geometry for catalysis to occur. In here we propose that the tryptophan at position 573 is important for the catalytic process.

• 미생물학회지
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• 제43권1호
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• pp.19-22
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• 2007

Acetohydroxylacid synthase (E.C.2.2.1.6.,AHAS)는 박테리아, 곰팡이, 식물 등에서 필수 아미노산중 세 가지 아미노산(Val, Leu, Ile)의 생합성에 관여하는 효소중 하나이다. Haemophilus influenzae에 대한 AHAS의 효소특성을 규명하기 위하여 H. influenzae의 AHAS catalytic subunit 유전자(TIGR access code HI2585)를 pET28a 발현 벡터에 삽입시켰고, 대장균 BL21(DE3)에서 C-말단에 일련의 histidine을 갖는 재조합 단백질로 발현시켰고, Histidine-tag affinity chromatography 및 gel filtration chromatography를 이용하여 단일 단백질로 정제하였다. 정제하여 얻은 단백질은 최대 15 mg/ml까지 농축이 가능하였다. 정제된 단백질의 분자량은 SDS-PAGE 전기 영동법을 이용하여 약 63.9 kDa의 분자량을 확인하였다. AHAS 효소 활성은 discontinuous colorimetric assay방법을 이용하여 측정하였다. H. influenzae AHAS catalytic subunit의 specific activity는 3.22 U/mg 이었다. 또한AHAS의 최적 활성 온도와 pH는 각각$37^{\circ}C$와 pH 7.5이었다. AHAS 효소 활성은buffer의 종류에 따라 차이가 있었으며, 유기용매가 증가함에 따라 효소 활성도 감소하였다.