title: Arginine 26 and aspartic acid 69 of the regulatory subunit are key residues of subunits interaction of acetohydroxyacid synthase isozyme III from E. coli.
abstract: Acetohydroxyacid synthase (AHAS), which catalyzes the first step in the biosynthesis of branched-chain amino acids, is composed of catalytic and regulatory subunits. The enzyme exhibits full activity only when the regulatory subunit (RSU) binds to the catalytic subunit (CSU). However, the crystal structure of the holoenzyme has not been reported yet, and the molecular interaction between the CSU and RSU is also unknown. Herein, we introduced a global-surface, site-directed labeling scanning method to determine the potential interaction region of the RSU. This approach relies on the insertion of a bulky fluorescent probe at the designated site on the surface of the RSU to cause a dramatic change in holoenzyme activity by perturbing subunit interaction. Then, the key amino acid residues in the potential interaction regions were identified by site-directed mutagenesis. Compared to the wild-type, the single-point mutants R26A and D69A showed 54 and 64 % activity, respectively, whereas the double mutant (R26A+D69A) gave 14 %, thus suggesting that residues Arg26 and Asp69 are the key residues of subunit interaction with cooperative action. Additionally, the results of GST pull-down assays and pH-dependence experiments suggested that polar interaction is the main force for subunits interaction. A plausible protein-protein interaction model of the holoenzyme of Escherichia coli AHAS III is proposed, based on the mutagenesis and protein docking studies. The protocol established here should be useful for the identification of the molecular interactions between proteins.
authors.affiliation: Department of Chemical Biology and State Key Laboratory of Elemento-organic Chemistry, Nankai University, Tianjin, 300071, China.
authors.forename: Yuefang
authors.initials: Y
authors.lastname: Zhao
authors.forename: Xin
authors.initials: X
authors.lastname: Wen
authors.forename: Congwei
authors.initials: C
authors.lastname: Niu
authors.forename: Zhen
authors.initials: Z
authors.lastname: Xi
mesh_terms: D000095:Acetolactate Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D021122:Protein Subunits; D001224:Aspartic Acid; D001120:Arginine; D000095:Acetolactate Synthase