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 °C, potassium phosphate buffer, 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 µM respectively. Inhibition of purified AHAS I protein was determined with herbicides and new compounds.
Department of Chemistry, Hanyang University, Seoul, Korea.
Ngoc Chien
NC
Pham
Ji-Young
JY
Moon
Jun-Haeng
JH
Cho
Soo-Jae
SJ
Lee
Joon-Shik
JS
Park
Dong-Eun
DE
Kim
Yoonkyung
Y
Park
Moon-Young
MY
Yoon
D000095:Acetolactate Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D000597:Amino Acids, Branched-Chain / Q000096:biosynthesis; D003001:Cloning, Molecular; D048168:Escherichia coli K12 / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006540:Herbicides / Q000494:pharmacology; D007700:Kinetics; D011485:Protein Binding; D013835:Thiamine Pyrophosphate / Q000378:metabolism
D000597:Amino Acids, Branched-Chain; D029968:Escherichia coli Proteins; D006540:Herbicides; D005182:Flavin-Adenine Dinucleotide; D000095:Acetolactate Synthase; C510092:IlvB protein, E coli; D013835:Thiamine Pyrophosphate