Mode of action of herbicidal ALS-inhibitors on acetolactate synthase from green plant cell cultures, yeast, and Escherichia coli

Inhibitors of branched-chain amino acid biosynthesis--by inhibiting acetolactate synthase (ALS)-represent the most active group of herbicidal compounds to date (Shaner, D.L., Recent Adv. Phytochem. 23 (1989) 227-61). A microbial screening technique has been developed to investigate known and possible new ALS-inhibitors. Escherichia coli mutant FD 1062, which expresses only valine-resistant ALS II isoenzyme as the solely branched-chain amino acid synthesizing isoenzyme, has been used extensively to optimize known and to screen for new chemical classes of ALS-inhibitors, respectively. Herbicidal compounds like sulfonylureas, triazolopyrimidines, pyrimidylsalicylates, carbamoylpyrazolines, sulfonylimino-azinyl-heteroazoles, sulfonylamide azines, and substituted sulfonyldiamides, respectively, are active on minimal medium with Ki-values which resemble the rank order of biological activity of these compounds in the greenhouse. Interestingly, herbicidal imidazolinones are not at all inhibitory on E. coli strain FD 1062 in vivo although, of course, they exert high activity on isolated bacterial ALS. Similarly, N-protected valylanilides, pyrimidyl mandelic acids, benzenesulfonyl carboxamides, and ubiquinone-O are inactive in the bacterial assay but have been shown by other methods to act as ALS inhibitors. Additionally, reversal experiments can he performed to exclude, e.g. artificial inhibitory effects of test compounds. Moreover, a thin-layer biogram application technique opens the opportunity to test mixtures of chemicals. From green plant cell cultures (Catharanthus roseus) ALS has been isolated and characterized in terms of inhibition by sulfonylureas, imidazolinones, triazolopyrimidines, salicylates, and carbamoylpyrazolines, respectively. All five types show biphasic slow tight binding kinetics with steady state I50 values of 0.5 nm (sulfometuron), 1.9 nm (triazolopyrimidine), 8.3 nm (salicylate), 23 nm (imazapyr), and 135 nm (carbamoylpyrazoline), respectively. Isolated ALS from Saccharomyces cerevisiae is equally well blocked by herbicidal ALS inhibitors although with different I50 values (triazolopyrimidine, 21 nm, sulfometuron, 70 nm, salicylate, 21 micromoles, imazapyr, 38 micromoles, and carbamoylpyrazoline, 148 micromoles). Surprisingly, biphasic kinetics could not be observed with the yeast enzyme although slow binding behaviour was clearly established.