title: Mutagenesis of Escherichia coli acetohydroxyacid synthase isoenzyme II and characterization of three herbicide-insensitive forms.
abstract: Sulphonylurea and imidazolinone herbicides act by inhibiting acetohydroxyacid synthase (AHAS; EC 4.1.3.18), the enzyme that catalyses the first step in the biosynthesis of branched-chain amino acids. AHAS requires as cofactors thiamin diphosphate, a bivalent metal ion and, usually, FAD. Escherichia coli contains three isoenzymes and this study concerns isoenzyme II, the most herbicide-sensitive of the E. coli forms. A plasmid containing the large and small subunit genes of AHAS II was mutagenized using hydroxylamine and clones resistant to the sulphonylurea chlorimuron ethyl were selected. Three mutants were isolated; A26V, V99M and A108V. A26V has been described previously whereas the equivalent mutation of A108V has been reported in a herbicide-insensitive variant of yeast AHAS. The V99M mutation has not been discovered previously in AHAS from any source. The mutants were each over-expressed in E. coli, and the enzymes were purified to homogeneity. Some differences from wild type in the kinetic properties (kcat, Km and cofactor affinities) were observed, most notably a 28-fold decrease in the affinity for thiamin diphosphate of V99M. None of the mutants shows marked changes from the wild type in sensitivity to three imidazolinones, with the largest increase in the apparent inhibition constant being a factor of approximately 5. The A26V mutant is weakly resistant (6- to 20-fold) to six sulphonylureas, whereas stronger resistance is seen in V99M (20- to 250-fold) and A108V (35- to 420-fold). Resistance as a result of these mutations is consistent with a molecular model of the herbicide-binding site, which predicts that mutation of G249 might also confer herbicide insensitivity. Three G249 mutants were constructed, expressed and purified but all are inactive, apparently because they cannot bind FAD.
authors.affiliation: Centre for Protein Structure, Function and Engineering, Department of Biochemistry, University of Queensland, Brisbane, Qld 4072, Australia.
authors.forename: C M
authors.initials: CM
authors.lastname: Hill
authors.forename: R G
authors.initials: RG
authors.lastname: Duggleby
mesh_terms: D000095:Acetolactate Synthase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D005798:Genes, Bacterial / Q000187:drug effects; D006540:Herbicides / Q000737:chemistry / Q000494:pharmacology*; D019811:Hydroxylamine / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013453:Sulfonylurea Compounds / Q000494:pharmacology
chemical_list: D006540:Herbicides; D007093:Imidazoles; D007527:Isoenzymes; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013453:Sulfonylurea Compounds; D019811:Hydroxylamine; D000095:Acetolactate Synthase