title: Functional evaluation of residues in the herbicide-binding site of Mycobacterium tuberculosis acetohydroxyacid synthase by site-directed mutagenesis.
abstract: Mycobacterium tuberculosis acetohydroxyacid synthase (M. tuberculosis AHAS) has been proposed to bean essential target for novel herbicide- and chemical-based antibacterial agents. Therefore, here we investigated the roles of multiple conserved herbicide-binding site residues (R318, A146, Q148, M512, and V513) in M. tuberculosis AHAS through site-directed mutagenesis by characterizing the kinetic parameters and herbicide sensitivities of various point mutants. Interestingly, all mutant enzymes showed significantly altered kinetic parameters, specifically reduced affinity towards both the substrate and cofactor. Importantly, mutation of R318 led to a complete loss of AHAS activity, indicating a key role for this residue in substrate binding. Furthermore, all mutants demonstrated significant herbicide resistance against chlorimuron ethyl (CE), with several-fold higher IC50 than that of wild type AHAS. Docking analysis also indicated that binding of CE was slightly affected upon mutation of these residues. Taken together, these data suggest that the residues examined here mediate CE binding and may also be important for the catalytic activity of AHAS. This study will pave the way for future structure-function studies of CE and will also aid the development of novel anti-tuberculosis agents based on this chemical scaffold.
authors.affiliation: Department of Chemistry, Institute of Natural Science, Hanyang University, Seoul 133-791, Republic of Korea.
authors.forename: In-Pil
authors.initials: IP
authors.lastname: Jung
authors.affiliation: Department of Chemistry, Institute of Natural Science, Hanyang University, Seoul 133-791, Republic of Korea.
authors.forename: Jun-Haeng
authors.initials: JH
authors.lastname: Cho
authors.affiliation: Department of Agricultural Biotechnology, National Academy of Agricultural Science, Jeonju 560-500, Republic of Korea.
authors.forename: Bon-Sung
authors.initials: BS
authors.lastname: Koo
authors.affiliation: Department of Chemistry, Institute of Natural Science, Hanyang University, Seoul 133-791, Republic of Korea. Electronic address: myyoon@hanyang.ac.kr.
authors.forename: Moon-Young
authors.initials: MY
authors.lastname: Yoon
mesh_terms: D000095:Acetolactate Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000995:Antitubercular Agents / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D006540:Herbicides / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D006540:Herbicides; D011994:Recombinant Proteins; D000095:Acetolactate Synthase
keywords: Acetohydroxyacid Synthase; Chlorimuron Ethyl; Herbicide-Binding Site; Site-Directed mutagenesis