Title
Point mutation (R153H or R153C) in Escherichia coliisocitrate dehydrogenase: Biochemical characterization and functional implication
Author
Ping Song, Shan Li, Yatao Wu, Changqi Lv, Peng Wang, Guoping Zhu
Year
2016
Journal
Journal of Basic Microbiology
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 Km values 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−1 s−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.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Biochemistry