Structural and functional evaluation of three well-conserved serine residues in tobacco acetohydroxyacid synthase.
The first step in the common pathway for the biosynthesis of branched-chain amino acids (BCAAs) is catalyzed by acetohydroxyacid synthase (AHAS). The roles of three well-conserved serine residues (S167, S506, and S539) in tobacco AHAS were determined using site-directed mutagenesis. The mutations S167F and S506F were found to be inactive and abolished the binding affinity for cofactor FAD. The Far-UV CD spectrum of the inactive mutants was similar to that of wild-type enzyme, indicating no major conformational changes in the secondary structure. However, the active mutants, S167R, S506A, S506R, S539A, S539F and S539R, showed lower specific activities. Further, a homology model of tobacco AHAS was generated based on the crystal structure of yeast AHAS. In the model, the S167 and S506 residues were identified near the FAD binding site, while the S539 residue was found to near the ThDP binding site. The S539 mutants, S539A and S539R, showed strong resistance to three classes of herbicides, NC-311 (a sulfonylurea), Cadre (an imidazolinone), and TP (a triazolopyrimidine). In contrast, the active S167 and S506 mutants did not show any significant resistance to the herbicides, with the exception of S506R, which showed strong resistance to all herbicides. Thus, our results suggest that the S167 and S506 residues are essential for catalytic activity by playing a role in the FAD binding site. The S539 residue was found to be near the ThDP with an essential role in the catalytic activity and specific mutants of this residue (S539A and S539R) showed strong herbicide resistance as well.
Yoon
Moon-Young
MY
Department of Chemistry, Hanyang University, Seoul, Republic of Korea. myyoon@hanyang.ac.kr
Gedi
Vinayakumar
V
Kim
Joungmok
J
Park
Yoonkyung
Y
Kim
Dong-Eun
DE
Park
Eun-Hye
EH
Choi
Jung-Do
JD
D000095:Acetolactate Synthase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D055162:Biocatalysis; D003067:Coenzymes / Q000494:pharmacology; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004789:Enzyme Activation / Q000187:drug effects; D006540:Herbicides / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry* / Q000378:metabolism*; D014026:Nicotiana / Q000201:enzymology*
D003067:Coenzymes; D006540:Herbicides; D012694:Serine; D000095:Acetolactate Synthase