Expression in Escherichia coli of a Putative Human Acetohydroxyacid Synthase

Duggleby, Ronald G.,Kartikasari, Apriliana E.R.,Wunsch, Rebecca M.,Lee, Yu-Ting,Kil, Mee-Wha,Shin, Ju-Young,Chang, Soo-Ik Korean Society for Biochemistry and Molecular Biol 2000 Journal of biochemistry and molecular biology Vol.33 No.3

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.

Expression in Escherichia coli of a Putative Human Acetohydroxyacid Synthase

Chang,Soo-Ik,Kartikasari,Apriliana E.R.,Wunsch,Rebecca M.,Lee,Yu-Ting,Kil,Mee-Wha,Shin,Ju-Young,Duggleby,Ronald G. The Korea Science and Technology Center 2000 BMB Reports Vol.33 No.3

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. Attempt 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.

Development of ssDNA aptamers as potent inhibitors of Mycobacterium tuberculosis acetohydroxyacid synthase.

Baig, Irshad Ahmed,Moon, Ji-Young,Lee, Sang-Choon,Ryoo, Sung-Weon,Yoon, Moon-Young Elsevier Science Publishers B.V 2015 Biochimica et biophysica acta. Proteins and proteo Vol.1854 No.10

<P>Acetohydroxyacid synthase (AHAS) from Mycobacterium tuberculosis (Mtb) is a promising potential drug target for an emerging class of new anti-tuberculosis agents. In this study, we identify short (30-mer) single-stranded DNA aptamers as a novel class of potent inhibitors of Mtb-AHAS through an in vitro DNA-SELEX method. Among all tested aptamers, two candidate aptamers (Mtb-Apt1 and Mtb-Apt6) demonstrated the greatest inhibitory potential against Mtb-AHAS activity with IC50 values in the low nanomolar range (28.940.002 and 22.350.001nM respectively). Interestingly, inhibition kinetics analysis of these aptamers showed different modes of enzyme inhibition (competitive and mixed type of inhibition respectively). Secondary structure-guided mutational modification analysis of Mtb-Apt1 and Mtb-Apt6 identified the minimal region responsible for their inhibitory action and consequently led to 17-mer and 20-mer shortened aptamers that retained equivalent or greater inhibitory potential. Notably, a modeling and docking exercise investigated the binding site of these two potent inhibitory aptamers on the target protein and showed possible involvement of some key catalytic dimer interface residues of AHAS in the DNA-protein interactions that lead to its potent inhibition. Importantly, these two short candidate aptamers, Mtb-Apt1 (17-mer) and Mtb-Apt6 (20-mer), also demonstrated significant growth inhibition against multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of tuberculosis with very low MIC of 5.36μg/ml and 6.24μg/ml, respectively and no significant cytotoxicity against mammalian cell line. This is the first report of functional inhibitory aptamers against Mtb-AHAS and provides the basis for development of these aptamers as novel and strong anti-tuberculosis agents.</P>

Haemophilus influenzae의 Acetohydroxyacid Synthase Catalytic Subunit 재조합 단백질 발현 및 특성

노경미,최경재,박준식,윤문영,Noh, Kyoung-Mi,Choi, Kyoung-Jae,Park, Joon-Shik,Yoon, Moon-Young 한국미생물학회 2007 미생물학회지 Vol.43 No.1

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. Acetohydroxyacid synthase (E.C.2.2.1.6., AHAS) is the enzyme that catalyses the first step in the synthesis of the branched-chain amino acids valine, leucine and isoleucine. The AHAS gene (TIGR access code HI2585) from Heamophilus influenzae was cloned into the bacterial expression vector pET-28a and expressed in the Escherichia coli strain BL21(DE3). The expressed enzyme was purified by $Ni^{2+}-charged$ HiTrap chelating HP column. The purified enzyme appears as a single band on SDS-PAGE with a molecular mass of about 63.9 kDa. The enzyme exhibits absolute dependence on the three cofactors FAD, $MgCl_{2}$ and thiamine diphosphate for activity. Specific activity of purified enzyme has 3.22 unit/mg and optimum activity in the pH 7.5 at $37^{\circ}C$. This enzyme activity has an effect on the buffer. When comparing the enzyme activity against the organic solvent, it followed in type and the difference it is but even from the aqueous solution where the organic solvent is included with the fact that the enzyme activity is maintained.

Characterization of Acetohydroxyacid Synthase I from <i>Escherichia coli</i> K-12 and Identification of Its Inhibitors

CHIEN, Pham Ngoc,MOON, Ji-Young,CHO, Jun-Haeng,LEE, Soo-Jae,PARK, Joon-Shik,KIM, Dong-Eun,PARK, Yoonkyung,YOON, Moon-Young Japan Society for Bioscience, Biotechnology, and A 2010 Bioscience, Biotechnology, and Biochemistry Vol.74 No.11

<P>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). <I>Escherichia coli</I> contains three active isoenzymes, and acetohydroxyacid synthase I (AHAS I) large subunit is encoded by the <I>ilvB</I> gene. In this study, the <I>ilvB</I> gene from <I>E. coli</I> K-12 was cloned into expression vector pETDuet-1, and was expressed in <I>E. coli</I> 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 <I>E. coli</I> K-12 AHAS I were 37 °C, potassium phosphate buffer, and 7.5. Km values for <I>E. coli</I> K-12 AHAS I binding to pyruvate, Mg<SUP>+2</SUP>, ThDP, and FAD were 4.15, 1.26, 0.2 m<SMALL>M</SMALL>, and 0.61 μ<SMALL>M</SMALL> respectively. Inhibition of purified AHAS I protein was determined with herbicides and new compounds.</P>

Structure and Functional Effect of Tryptophan Mutants of Nicotiana tabacum Acetohydroxyacid Synthase

윤문영,마수드카림,라임정 대한화학회 2008 Bulletin of the Korean Chemical Society Vol.29 No.9

Characterization of acetohydroxyacid synthase from <i>Mycobacterium tuberculosis</i> and the identification of its new inhibitor from the screening of a chemical library

Choi, Kyoung-Jae,Yu, Yeon Gyu,Hahn, Hoh Gyu,Choi, Jung-Do,Yoon, Moon-Young Elsevier 2005 FEBS letters Vol.579 No.21

<P><B>Abstract</B></P><P>Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate- (ThDP-) and FAD-dependent enzyme that catalyzes the first common step in the biosynthetic pathway of the branched-amino acids such as leucine, isoleucine, and valine. The genes of AHAS from <I>Mycobacterium tuberculosis</I> were cloned, and overexpressed in <I>E. coli</I> and purified to homogeneity. The purified AHAS from <I>M. tuberculosis</I> is effectively inhibited by pyrazosulfuron ethyl (PSE), an inhibitor of plant AHAS enzyme, with the IC<SUB>50</SUB> (inhibitory concentration 50%) of 0.87μM. The kinetic parameters of <I>M. tuberculosis</I> AHAS were determined, and an enzyme activity assay system using 96-well microplate was designed. After screening of a chemical library composed of 5600 compounds using the assay system, a new class of AHAS inhibitor was identified with the IC<SUB>50</SUB> in the range of 1.8–2.6μM. One of the identified compounds (KHG20612) further showed growth inhibition activity against various strains of <I>M. tuberculosis</I>. The correlation of the inhibitory activity of the identified compound against AHAS to the cell growth inhibition activity suggested that AHAS might be served as a target protein for the development of novel anti-tuberculosis therapeutics.</P>

Mechanism Studies of Substituted Triazol-1-yl-pyrimidine Derivatives Inhibition on Mycobacterium tuberculosis Acetohydroxyacid Synthase

Chien, Pham Ngoc,Jung, In-Pil,Reddy, Katta Venugopal,Yoon, Moon-Young Korean Chemical Society 2012 Bulletin of the Korean Chemical Society Vol.33 No.12

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 $2.24{\AA}$.

Sulfonylurea is a Non-competitive Inhibitor of Acetohydroxyacid Synthase from Mycobacterium tuberculosis

최경재,노경미,최정도,박전식,원호식,김정림,김정선,윤문영 대한화학회 2006 Bulletin of the Korean Chemical Society Vol.27 No.10

Characterization of the Catalytic Properties of Recombinant Acetohydroxyacid Synthase from Tobacco

김정목,최정도,김복환,윤문영 대한화학회 2005 Bulletin of the Korean Chemical Society Vol.26 No.2

The nature of the active site of Tobacco acetohydroxyacid synthase (AHAS) in the substrate- and cofactor-binding was studied by kinetics and fluorescence spectroscopy. The substrate saturation curve does not follow Michaelis-Menten kinetics at different temperatures (7, 21 and 37 oC), pH (6.5, 7.5 and 8.5) and buffers (Tris-HCl and MOPS). The concentration of one half of the maximum velocity (S0.5) decreased in the following order: pyruvate > ThDP Mg+2 > FAD. However, the catalytic efficiency (Kcat/S0.5) inversely decreased in the following order; FAD > Mg+2 ThDP > pyruvate, indicating that the cofactors by in decreasing order; FAD, Mg+2, ThDP, affect the catalysis of AHAS. The dissociation constant (Kd) of the intrinsic tryptophan fluorescence decreased with the same tendency of the concentration of one half of the maximum velocity (S0.5) 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.