HERBICIDE-RESISTANT FORMS OF ARABIDOPSIS-THALIANA ACETOHYDROXYACID SYNTHASE - CHARACTERIZATION OF THE CATALYTIC PROPERTIES AND SENSITIVITY TO INHIBITORS OF 4 DEFINED MUTANTS

Acetohydroxyacid synthase (AHAS) catalyses the first step in the synth esis of the branched-chain amino acids and is the target of several cl asses of herbicides. Four mutants (A122V, W574S, W574L and S653N) of t he AHAS gene from Arabidopsis thaliana were constructed, expressed in Escherichia coli, and the enzymes were purified. Each mutant form and wild-type was characterized with respect to its catalytic properties a nd sensitivity to nine herbicides. Each enzyme had a pH optimum near 7 .5. The specific activity varied from 13 % (A122V) to 131 % (W574L) of the wild-type and the K-m for pyruvate of the mutants was similar to the wild-type, except for W574L where it was five-fold higher. The act ivation by cofactors (FAD, Mg2+ and thiamine diphosphate) was examined . A122V showed reduced affinity for all three cofactors, whereas S653N bound FAD more strongly than wild-type AHAS. Six sulphonylurea herbic ides inhibited A122V to a similar degree as the wild-type but S653N sh owed a somewhat greater reduction in sensitivity to these compounds. I n contrast, the W574 mutants were insensitive to these sulphonylureas, with increases in the K-i(app) (apparent inhibition constant) of seve ral hundred fold. All four mutants were resistant to three imidazolino ne herbicides with decreases in sensitivity ranging from 100-fold to m ore than 1000-fold.