Roles of lysine 219 and 255 residues in tobacco acetolactate synthase.
Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. The ALS is the target of several classes of herbicides, including the sulfonylureas, the imidazolinones, and the triazolopyrimidines. The roles of three well-conserved lysine residues (K219, K255, K299) in tobacco ALS were determined using site-directed mutagenesis. The mutation of K219Q inactivated the enzyme and abolished the binding affinity for cofactor FAD. However, the secondary structure of the enzyme was not changed significantly by the mutation. Both mutants, K255F and K255Q, showed strong resistance to three classes of herbicides Londax (a sulfonylurea), Cadre (an imidazolinone), and TP (a triazolopyrimidine). In addition, there was no difference in the secondary structures of wALS and K255F. On the other hand, the mutation of K299Q did not show any significant effect on the kinetic properties or any sensitivity to the herbicides. These results suggest that Lys219 is located at the active site and is likely involved in the binding of FAD, and that Lys255 is located at a binding site common for the three herbicides in tobacco ALS.
Yoon
Tae-Yeol
TY
School of Life Science and Research Institute for Genetic Engineering, Chungbuk National University, Cheongju 361-763, Republic of Korea.
Chung
Sun-Mee
SM
Chang
Soo-Ik
SI
Yoon
Moon-Young
MY
Hahn
Tae-Ryong
TR
Choi
Jung-Do
JD
D000095:Acetolactate Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006540:Herbicides / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D014026:Nicotiana / Q000201:enzymology*
D004791:Enzyme Inhibitors; D006540:Herbicides; D011994:Recombinant Proteins; D000095:Acetolactate Synthase; D008239:Lysine