Expression of Acetohydroxyacid Synthase from Bacillus anthracis and Its Potent Inhibitors

Choi, Kyoung‐Jae; Pham, Chien Ngoc; Jung, Hoeil; Han, Sung‐Hwan; Choi, Jung‐Do; Kim, Jinheung; Yoon, Moon‐Young

doi:10.5012/bkcs.2007.28.7.1109

Bulletin of the Korean Chemical Society

2007

DOI: 10.5012/bkcs.2007.28.7.1109

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Expression of Acetohydroxyacid Synthase from Bacillus anthracis and Its Potent Inhibitors

Kyoung‐Jae Choi¹,

Chien Ngoc Pham²,

Hoeil Jung³

et al.

Abstract: 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 wa… Show more

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Cited by 9 publications

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Arginine 26 and Aspartic Acid 69 of the Regulatory Subunit are Key Residues of Subunits Interaction of Acetohydroxyacid Synthase Isozyme III from E. coli

et al. 2012

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Acetohydroxyacid synthase (AHAS), which catalyzes the first step in the biosynthesis of branched-chain amino acids, is composed of catalytic and regulatory subunits. The enzyme exhibits full activity only when the regulatory subunit (RSU) binds to the catalytic subunit (CSU). However, the crystal structure of the holoenzyme has not been reported yet, and the molecular interaction between the CSU and RSU is also unknown. Herein, we introduced a global-surface, site-directed labeling scanning method to determine the potential interaction region of the RSU. This approach relies on the insertion of a bulky fluorescent probe at the designated site on the surface of the RSU to cause a dramatic change in holoenzyme activity by perturbing subunit interaction. Then, the key amino acid residues in the potential interaction regions were identified by site-directed mutagenesis. Compared to the wild-type, the single-point mutants R26A and D69A showed 54 and 64 % activity, respectively, whereas the double mutant (R26A+D69A) gave 14 %, thus suggesting that residues Arg26 and Asp69 are the key residues of subunit interaction with cooperative action. Additionally, the results of GST pull-down assays and pH-dependence experiments suggested that polar interaction is the main force for subunits interaction. A plausible protein-protein interaction model of the holoenzyme of Escherichia coli AHAS III is proposed, based on the mutagenesis and protein docking studies. The protocol established here should be useful for the identification of the molecular interactions between proteins.

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Arginine 26 and Aspartic Acid 69 of the Regulatory Subunit are Key Residues of Subunits Interaction of Acetohydroxyacid Synthase Isozyme III from E. coli

et al. 2012

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The Minimum Activation Peptide from ilvH Can Activate the Catalytic Subunit of AHAS from Different Species

et al. 2013

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Acetohydroxyacid synthases (AHASs), which catalyze the first step in the biosynthesis of branched-chain amino acids, are composed of a catalytic subunit (CSU) and a regulatory subunit (RSU). The CSU harbors the catalytic site, and the RSU is responsible for the activation and feedback regulation of the CSU. Previous results from Chipman and co-workers and our lab have shown that heterologous activation can be achieved among isozymes of Escherichia coli AHAS. It would be interesting to find the minimum peptide of ilvH (the RSU of E. coli AHAS III) that could activate other E. coli CSUs, or even those of ## species. In this paper, C-terminal, N-terminal, and C- and N-terminal truncation mutants of ilvH were constructed. The minimum peptide to activate ilvI (the CSU of E. coli AHAS III) was found to be ΔN 14-ΔC 89. Moreover, this peptide could not only activate its homologous ilvI and heterologous ilvB (CSU of E. coli AHAS I), but also heterologously activate the CSUs of AHAS from Saccharomyces cerevisiae, Arabidopsis thaliana, and Nicotiana plumbaginifolia. However, this peptide totally lost its ability for feedback regulation by valine, thus suggesting different elements for enzymatic activation and feedback regulation. Additionally, the apparent dissociation constant (Kd ) of ΔN 14-ΔC 89 when binding CSUs of different species was found to be 9.3-66.5 μM by using microscale thermophoresis. The ability of this peptide to activate different CSUs does not correlate well with its binding ability (Kd ) to these CSUs, thus implying that key interactions by specific residues is more important than binding ability in promoting enzymatic reactions. The high sequence similarity of the peptide ΔN 14-ΔC 89 to RSUs across species hints that this peptide represents the minimum activation motif in RSU and that it regulates all AHASs.

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Evaluation of substituted triazol-1-yl-pyrimidines as inhibitors of Bacillus anthracis acetohydroxyacid synthase

Gedi

Jayaraman

Kalme

et al. 2010

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Expression of Acetohydroxyacid Synthase from Bacillus anthracis and Its Potent Inhibitors

Cited by 9 publications

References 9 publications

Arginine 26 and Aspartic Acid 69 of the Regulatory Subunit are Key Residues of Subunits Interaction of Acetohydroxyacid Synthase Isozyme III from E. coli

Arginine 26 and Aspartic Acid 69 of the Regulatory Subunit are Key Residues of Subunits Interaction of Acetohydroxyacid Synthase Isozyme III from E. coli

The Minimum Activation Peptide from ilvH Can Activate the Catalytic Subunit of AHAS from Different Species

Evaluation of substituted triazol-1-yl-pyrimidines as inhibitors of Bacillus anthracis acetohydroxyacid synthase

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