title: Characterization of Aspartate Kinase from Corynebacterium pekinense and the Critical Site of Arg169.
abstract: Aspartate kinase (AK) is the key enzyme in the biosynthesis of aspartate-derived amino acids. Recombinant AK was efficiently purified and systematically characterized through analysis under optimal conditions combined with steady-state kinetics study. Homogeneous AK was predicted as a decamer with a molecular weight of ~48 kDa and a half-life of 4.5 h. The enzymatic activity was enhanced by ethanol and Ni(2+). Moreover, steady-state kinetic study confirmed that AK is an allosteric enzyme, and its activity was inhibited by allosteric inhibitors, such as Lys, Met, and Thr. Theoretical results indicated the binding mode of AK and showed that Arg169 is an important residue in substrate binding, catalytic domain, and inhibitor binding. The values of the kinetic parameter Vmax of R169 mutants, namely, R169Y, R169P, R169D, and R169H AK, with l-aspartate as the substrate, were 4.71-, 2.25-, 2.57-, and 2.13-fold higher, respectively, than that of the wild-type AK. Furthermore, experimental and theoretical data showed that Arg169 formed a hydrogen bond with Glu92, which functions as the entrance gate. This study provides a basis to develop new enzymes and elucidate the corresponding amino acid production.
authors.affiliation: College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China. minwh2000@jlau.edu.cn.|National Engineering Laboratory of Wheat and Corn Deep Processing, Changchun 130118, China. minwh2000@jlau.edu.cn.
authors.forename: Weihong
authors.initials: W
authors.lastname: Min
authors.affiliation: College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China. lihuiying@jlau.edu.cn.|National Engineering Laboratory of Wheat and Corn Deep Processing, Changchun 130118, China. lihuiying@jlau.edu.cn.
authors.forename: Huiying
authors.initials: H
authors.lastname: Li
authors.affiliation: College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China. lihongmei@jlau.edu.cn.|National Engineering Laboratory of Wheat and Corn Deep Processing, Changchun 130118, China. lihongmei@jlau.edu.cn.
authors.forename: Hongmei
authors.initials: H
authors.lastname: Li
authors.affiliation: College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China. liuchunlei0709@jlau.edu.cn.|National Engineering Laboratory of Wheat and Corn Deep Processing, Changchun 130118, China. liuchunlei0709@jlau.edu.cn.
authors.forename: Chunlei
authors.initials: C
authors.lastname: Liu
authors.affiliation: College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China. liujs2000@jlau.edu.cn.|National Engineering Laboratory of Wheat and Corn Deep Processing, Changchun 130118, China. liujs2000@jlau.edu.cn.
authors.forename: Jingsheng
authors.initials: J
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D001222:Aspartate Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D020134:Catalytic Domain; D003352:Corynebacterium / Q000201:enzymology* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins; D016415:Sequence Alignment; D012997:Solvents; D013816:Thermodynamics
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D012997:Solvents; D001222:Aspartate Kinase
keywords: Corynebacterium pekinense; aspartate kinase; characterization; molecular docking