title: Point mutation (R153H or R153C) in Escherichia coli isocitrate dehydrogenase: Biochemical characterization and functional implication.
abstract: Arginine 132 (R132) mutations to histidine or cysteine frequently occur to cytosolic NADP+-isocitrate dehydrogenase (IDH1) in secondary glioblastoma multiforme (GBM) patients, in which GBM develops from a lower grade astroctyoma. Mutant enzymes lose the normal IDH activity, but acquire a neomorphic ability of producing 2-hydroxyglutarate (2-HG) from α-ketoglutarate (α-KG). In the present study, the analogous mutations, Arg to His or Cys, were employed to homologous Arg153 of the NADP+-IDH from Escherichia coli (EcIDH), generating two mutants: EcIDH R153 H and EcIDH R153C. The mutations dramatically reduced the catalytic efficiencies (kcat/Km) of EcIDH R153H and EcIDH R153C for isocitrate oxidation, which dropped to only 0.6 and 1.5% of the wild-type enzyme, respectively. Neoenzymatic activities of catalyzing α-KG to 2-HG by EcIDH R153H and EcIDH R153C were confirmed by GC/TOF-MS analysis. The Kmvalues of EcIDH R153H and EcIDH R153C displayed for α-KG were 3.3 ± 0.12 and 2.2 ± 0.13 mM, respectively, and the catalytic efficiencies (kcat/Km) of the two mutants for α-KG were 300 and 450 M-1s-1, respectively. As human IDH1 Arg132 mutation is cancer-associated, the present study provides new information for the in-depth investigation of the metabolic influence of EcIDH Arg mutation in vivo.
authors.affiliation: The Research Center of Life Omics and Health, Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu, 241000, Anhui, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Song
authors.affiliation: The Research Center of Life Omics and Health, Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu, 241000, Anhui, China.
authors.forename: Shan
authors.initials: S
authors.lastname: Li
authors.affiliation: The Research Center of Life Omics and Health, Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu, 241000, Anhui, China.
authors.forename: Yatao
authors.initials: Y
authors.lastname: Wu
authors.affiliation: The Research Center of Life Omics and Health, Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu, 241000, Anhui, China.
authors.forename: Changqi
authors.initials: C
authors.lastname: Lv
authors.affiliation: The Research Center of Life Omics and Health, Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu, 241000, Anhui, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.affiliation: The Research Center of Life Omics and Health, Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu, 241000, Anhui, China.
authors.forename: Guoping
authors.initials: G
authors.lastname: Zhu
mesh_terms: D001120:Arginine / Q000235:genetics; D055162:Biocatalysis; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005977:Glutarates / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016415:Sequence Alignment
chemical_list: D005977:Glutarates; C019417:alpha-hydroxyglutarate; D006639:Histidine; D001120:Arginine; D007521:Isocitrate Dehydrogenase; D003545:Cysteine
keywords: Arginine mutation; Escherichia coli; GC; Isocitrate dehydrogenase; Neoenzymatic activity; TOF-MS