title: Molecular cloning, purification, and biochemical characterization of recombinant isocitrate dehydrogenase from Streptomyces coelicolor M-145.
abstract: Isocitrate dehydrogenase is a key enzyme in carbon metabolism. In this study we demonstrated that SCO7000 of Streptomyces coelicolor M-145 codes for the isocitrate dehydrogenase. Recombinant enzyme expressed in Escherichia coli had a specific activity of 25.3 μmoles/mg/min using NADP(+) and Mn(2+) as a cofactor, 40-times higher than that obtained in cell-free extract. Pure IDH showed a single band with an apparent Mr of 84 KDa in SDS-PAGE, which was also recognized as His-tag protein in the Western blot. Unexpectedly, in ND-PAGE conditions showed a predominant band of ~168 KDa that corresponded to the dimeric form of ScIDH. Also, zymogram assay and analytical gel filtration reveal that dimer was the active form. Kinetic parameters were 1.38, 0.11, and 0.109 mM for isocitrate, NADP, and Mn(2+), respectively. ATP, ADP, AMP, and their mixtures were the main ScIDH activity inhibitors. Zn(2+), Mg(2+), Ca(2+), and Cu(+) had inhibitory effect on enzyme activity.
authors.affiliation: a Departamento de Biología Molecular y Biotecnología , Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México , México, D.F., México.
authors.forename: Tóshiko
authors.initials: T
authors.lastname: Takahashi-Iñiguez
authors.forename: Saul
authors.initials: S
authors.lastname: Cruz-Rabadán
authors.forename: Luis Miguel
authors.initials: LM
authors.lastname: Burciaga-Cifuentes
authors.forename: María Elena
authors.initials: ME
authors.lastname: Flores
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D007521:Isocitrate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D007523:Isocitrates / Q000378:metabolism; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000235:genetics
chemical_list: D007523:Isocitrates; D011994:Recombinant Proteins; C034219:isocitric acid; D007521:Isocitrate Dehydrogenase
keywords: Streptomyces coelicolor; carbon metabolism; dimeric isocitrate dehydrogenase; tricarboxylic acid cycle