Characterization of Acetohydroxyacid Synthase I from Escherichia coli K-12 and Identification of Its Inhibitors

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 degrees C, potassium phosphate butler, and 7.5.. Km values for E. coli K-12 AHAS I binding to pyruvate, Mg+2, ThDP, and FAD were 4.15, 1.26, 0.2 mM, and 0.61 mu M respectively. Inhibition of purified AHAS I protein was determined with herbicides and new compounds.This work was supported by a Korea Research Foundation Grant (KRF-2008-313-000530) and by the Nuclear R&D program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology