MUTAGENESIS OF ESCHERICHIA-COLI ACETOHYDROXYACID SYNTHASE ISOENZYME-II AND CHARACTERIZATION OF 3 HERBICIDE-INSENSITIVE FORMS

Sulphonylurea and imidazolinone herbicides act by inhibiting acetohydr oxyacid synthase (AHAS; EC 4.1.3.18), the enzyme that catalyses the fi rst step in the biosynthesis of branched-chain amino acids. AHAS requi res as cofactors thiamin diphosphate, a bivalent metal ion and, usuall y, FAD. Escherichia coli contains three isoenzymes and this study conc erns 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 sulphonylu rea chlorimuron ethyl were selected. Three mutants were isolated; A26V , V99M and A108V. A26V has been described previously whereas the equiv alent mutation of A108V has been reported in a herbicide-insensitive v ariant of yeast AHAS. The V99M mutation has not been discovered previo usly in AHAS from any source. The mutants were each over-expressed in E. coli, and the enzymes were purified to homogeneity. Some difference s from wild type in the kinetic properties (k(eat), K-m and cofactor a ffinities) were observed, most notably a 28-fold decrease in the affin ity for thiamin diphosphate of V99M. None of the mutants shows marked changes from the wild type in sensitivity to three imidazolinones, wit h the largest increase in the apparent inhibition constant being a fac tor of approximately 5. The A26V mutant is weakly resistant (6- to 20- fold) to six sulphonylureas, whereas stronger resistance is seen in V9 9M (20- to 250-fold) and A108V (35- to 420-fold). Resistance as a resu lt of these mutations is consistent with a molecular model of the herb icide-binding site, which predicts that mutation of G249 might also co nfer herbicide insensitivity. Three G249 mutants were constructed, exp ressed and purified but all are inactive, apparently because they cann ot bind FAD.