Cm. Hill et Rg. Duggleby, MUTAGENESIS OF ESCHERICHIA-COLI ACETOHYDROXYACID SYNTHASE ISOENZYME-II AND CHARACTERIZATION OF 3 HERBICIDE-INSENSITIVE FORMS, Biochemical journal, 335, 1998, pp. 653-661
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.