Roles of conserved methionine residues in tobacco acetolactate synthase.
Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. ALS is the target of several classes of herbicides, including the sulfonylureas, the imidazolinones, and the triazolopyrimidines. The conserved methionine residues of ALS from plants were identified by multiple sequence alignment using ClustalW. The alignment of 17 ALS sequences from plants revealed 149 identical residues, seven of which were methionine residues. The roles of three well-conserved methionine residues (M350, M512, and M569) in tobacco ALS were determined using site-directed mutagenesis. The mutation of M350V, M512V, and M569V inactivated the enzyme and abolished the binding affinity for cofactor FAD. Nevertheless, the secondary structure of each of the mutants determined by CD spectrum was not affected significantly by the mutation. Both M350C and M569C mutants were strongly resistant to three classes of herbicides, Londax (a sulfonylurea), Cadre (an imidazolinone), and TP (a triazolopyrimidine), while M512C mutant did not show a significant resistance to the herbicides. The mutant M350C was more sensitive to pH change, while the mutant M569C showed a profile for pH dependence activity similar to that of wild type. These results suggest that M512 residue is likely located at or near the active site, and that M350 and M569 residues are probably located at the overlapping region between the active site and a common herbicide binding site.
Tien Le
Dung
D
School of Life Sciences, Chungbuk National University, Cheongju 361-763, Republic of Korea.
Yoon
Moon-Young
MY
Kim
Young Tae
YT
Choi
Jung-Do
JD
D000095:Acetolactate Synthase / Q000737:chemistry*; D000465:Algorithms; D001665:Binding Sites; D002942:Circular Dichroism; D019992:Databases as Topic; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D017343:Genes, Plant; D006540:Herbicides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008715:Methionine / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013053:Spectrophotometry; D014026:Nicotiana / Q000201:enzymology*
D006540:Herbicides; D005182:Flavin-Adenine Dinucleotide; D008715:Methionine; D000095:Acetolactate Synthase