Roles of histidine residues in tobacco acetolactate synthase

Acetolactate synthase (ALS) catalyzes the first common step ill the biosynt hesis of valine, leucine, and isoleucine in plants and microorganisms, ALS is the target of several structurally diverse classes of herbicides, includ ing sulfonylureas, imidazolinones, and triazolopyrimidines. The roles of th ree well-conserved histidine residues (H351, H392, and H487) in tobacco ALS were determined using site directed mutagenesis. Both H487F and H487L muta tions abolished the enzymatic activity as well as the binding affinity for the cofactor EAD. Nevertheless, the mutation of H487F did not affect the se condary structure of the ALS. The K-m values of H351M, H351Q, and H351F are approximately 18-, 60-, and fivefold higher than that of the wild-type ALS , respectively. Moreover, the K-c value of H351Q for FAD is about 137-fold higher than that of wALS. Mutants H351M and H351Q showed very strong resist ance to Londax (a sulfonylurea) and Cadre (an imidazolinone), whereas mutan t H351F was weakly resistant to them. However, the secondary structures of mutants H351M and H351Q appeared to be different from that of wALS. The mut ation of H392M did not have any significant effect on the kinetic parameter s nor the resistance to ALS-inhibiting herbicides. These results suggest th at the His487? residue is located at the active site of the enzyme and is l ikely involved in the binding of cofactor FAD in tobacco ALS, Mutational an alyses of the His351 residue imply that the active site of the ALS is proba bly close to its binding site of the herbicides, Londax and Cadre. (C) 2001 Academic Press.