title: Enzymatic characterization of a monomeric isocitrate dehydrogenase from Streptomyces lividans TK54.
abstract: Isocitrate dehydrogenase (IDH) is one of the key enzymes in the citric acid cycle, which involves in providing energy and biosynthetic precursors for metabolism. Here, we report for the first time the enzymatic characterization of a monomeric NADP(+)-dependent IDH from Streptomyces lividans TK54 (SlIDH). The icd gene (GenBank database accession number EU661252) encoding IDH was cloned and overexpressed in Escherichia coli. The molecular mass of SlIDH was about 80 kDa, typical of a monomeric NADP-IDH, and showed high amino acid sequence identity with known monomeric IDHs. The optimal activity of the 6His-tagged SlIDH was found at pH values 8.5 (Mn(2+)) and 9.0 (Mg(2+)), and the optimal temperature was around 46 degrees C. Heat-inactivation studies showed that about 50% SlIDH activity was preserved at 38 degrees C after 20 min of incubation. The recombinant SlIDH displayed a 62,000-fold (k(cat)/K(m)) preference for NADP(+) over NAD(+) with Mn(2+), and a 85,000-fold greater specificity for NADP(+) than NAD(+) with Mg(2+). Therefore, SlIDH is a divalent cation-dependent monomeric IDH with remarkably high coenzyme preference for NADP(+).
authors.affiliation: Key Laboratory of Molecular Evolution and Biodiversity and Institute of Molecular Biology and Biotechnology, College of Life Sciences, Anhui Normal University, Wuhu 241000, China.
authors.forename: Beibei
authors.initials: B
authors.lastname: Zhang
authors.forename: Baojuan
authors.initials: B
authors.lastname: Wang
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.forename: Zhengyu
authors.initials: Z
authors.lastname: Cao
authors.forename: Enqi
authors.initials: E
authors.lastname: Huang
authors.forename: Jiasheng
authors.initials: J
authors.lastname: Hao
authors.forename: Antony M
authors.initials: AM
authors.lastname: Dean
authors.forename: Guoping
authors.initials: G
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009249:NADP / Q000737:chemistry*; D016415:Sequence Alignment; D048170:Streptomyces lividans / Q000201:enzymology* / Q000235:genetics
chemical_list: D009249:NADP; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)