title: Biochemical Characterization and Complete Conversion of Coenzyme Specificity of Isocitrate Dehydrogenase from Bifidobacterium longum.
abstract: Bifidobacterium longum is a very important gram-positive non-pathogenic bacterium in the human gastrointestinal tract for keeping the digestive and immune system healthy. Isocitrate dehydrogenase (IDH) from B. longum (BlIDH), a novel member in Type II subfamily, was overexpressed, purified and biochemically characterized in detail. The active form of BlIDH was an 83-kDa homodimer. Kinetic analysis showed BlIDH was a NADP⁺-dependent IDH (NADP-IDH), with a 567- and 193-fold preference for NADP⁺ over NAD⁺ in the presence of Mg(2+) and Mn(2+), respectively. The maximal activity for BlIDH occurred at 60 °C (with Mn(2+)) and 65 °C (with Mg(2+)), and pH 7.5 (with Mn(2+)) and pH 8.0 (with Mg(2+)). Heat-inactivation profiles revealed that BlIDH retained 50% of maximal activity after incubation at 45 °C for 20 min with either Mn(2+) or Mg(2+). Furthermore, the coenzyme specificity of BlIDH can be completely reversed from NADP⁺ to NAD⁺ by a factor of 2387 by replacing six residues. This current work, the first report on the coenzyme specificity conversion of Type II NADP-IDHs, would provide better insight into the evolution of NADP⁺ use by the IDH family.
authors.affiliation: The Research Center of Life Omics and Health and Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu 241000, Anhui, China. huangsp2014@gmail.com.
authors.forename: Shi-Ping
authors.initials: SP
authors.lastname: Huang
authors.affiliation: The Research Center of Life Omics and Health and Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu 241000, Anhui, China. chengmei325111@163.com.
authors.forename: Hong-Mei
authors.initials: HM
authors.lastname: Cheng
authors.affiliation: The Research Center of Life Omics and Health and Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu 241000, Anhui, China. wangpeng1219@yahoo.com.
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.affiliation: The Research Center of Life Omics and Health and Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu 241000, Anhui, China. gpz1996@yahoo.com.
authors.forename: Guo-Ping
authors.initials: GP
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001644:Bifidobacterium / Q000201:enzymology* / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism*; D004795:Enzyme Stability; D007521:Isocitrate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D003067:Coenzymes; D009243:NAD; D008345:Manganese; D007521:Isocitrate Dehydrogenase; D008274:Magnesium
keywords: Bifidobacterium longum; biochemical characterization; coenzyme specificity determinants; isocitrate dehydrogenase; kinetics