Role of tryptophanyl residues in tobacco acetolactate synthase

Acetolactate synthase (ALS) catalyzes the first common step in the biosynth esis of valine, leucine, and isoleucine. ALS is the target of three classes of herbicides, the sulfonylureas, the imidazolinones, and the triazolopyri midines. Five mutants (W266F, W439F, W490F, W503F, and W573F) of the ALS ge ne from Nicotiana tabacum were constructed and expressed in Escherichia col i, and the enzymes were purified. The W490F mutation abolished the binding affinity for cofactor FAD and inactivated the enzyme. The replacement of Tr p573 by Phe yielded a mutant ALS resistant to the three classes of herbicid es. The other three mutations, W266F, W439F, and W503F, did not significant ly affect the enzymatic properties and the sensitivity to the herbicides. T hese results indicate that the Trp490 residue is essential for the binding of FAD and that Trp573 is located at the herbicide binding site. The data a lso suggest that the three classes of herbicides bind ALS competitively. (C ) 1999 Academic Press.