Arabidopsis acetohydroxyacid synthase expressed in Escherichia coli is insensitive to the feedback inhibitors

• Published in: Plant physiology (Bethesda) Vol. 99; no. 3; pp. 812 - 816
• Main Authors: Singh, B. (American Cyanamid Company, Princeton, NJ), Szamosi, I, Hand, J.M, Misra, R
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Physiologists 01.07.1992
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Analytical, structural and metabolic biochemistry; Biological and medical sciences; BIOSINTESIS; BIOSYNTHESE; Biosynthesis; Cell growth; Chloroplasts; CODE GENETIQUE; CODIGO GENETICO; CRUCIFERAE; Enzymes; Enzymes and enzyme inhibitors; ESCHERICHIA; Escherichia coli; EXPRESION GENICA; EXPRESSION DES GENES; Fundamental and applied biological sciences. Psychology; HERBICIDAS; HERBICIDE; Herbicides; INHIBIDORES DE ENZIMAS; INHIBITEUR D'ENZYME; ISOLEUCINA; ISOLEUCINE; LEUCINA; LEUCINE; LIGASAS; LIGASE; Lyases; Molecular Biology and Gene Regulation; Plants; Plasmids; Proteins; Seedlings; TRANSFORMACION GENETICA; TRANSFORMATION GENETIQUE; VALINA; VALINE; Genetic transformation; Enzyme; Escherichia coli; Biosynthesis; Gene expression; Herbicide; Arabidopsis thaliana; Cruciferae; Dicotyledones; Aminoacid; Angiospermae; Acetolactate synthase; Bacteria; Ureas; Spermatophyta; Inhibition; Molecular cloning; Enterobacteriaceae
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.99.3.812

Acetohydroxyacid synthase (AHAS), the first enzyme unique to the biosynthesis of isoleucine, leucine, and valine, is the target enzyme for several classes of herbicides. The AHAS gene from Arabidopsis thaliana including the chloroplast transit peptide, was cloned into the bacterial expression plasmid pKK233-2. The resulting plasmid was used to transform an AHAS-deficient Escherichia coli strain MF2000. The growth of the MF2000 strain of E. coli was complemented by the functional expression of the Arabidopsis AHAS. The AHAS protein was processed to a molecular mass of 65 kilodaltons that was similar to the mature protein isolated from Arabidopsis seedlings. The AHAS activity extracted from the transformed E. coli cells was inhibited by imidazolinone and sulfonylurea herbicides. AHAS activity extracted from Arabidopsis is inhibited by valine and leucine; however, this activity was insensitive to these feedback inhibitors when extracted from the transformed E. coli