title: Biochemical characterization of NADP⁺-dependent isocitrate dehydrogenase from Microcystis aeruginosa PCC7806.
abstract: Microcystis aeruginosa is the key symptom of water eutrophication and produces persistent microcystins. Our special attention was paid to the isocitrate dehydrogenase (IDH) of M. aeruginosa (MaIDH) because it plays important roles in energy and biosynthesis metabolisms and its catalytic product 2-oxoglutarate provides the carbon skeleton for ammonium assimilation and also constitutes a signaling molecule of nitrogen starvation in cyanobacteria. Sequence alignment showed that MaIDH shared significant sequence identity with IDHs from other cyanobacteria (>80 %) and other bacteria (>45 %). The subunit molecular weight of MaIDH was determined to be 52.6 kDa by filtration chromatography, suggesting MaIDH is a typical homodimer. The purified recombinant MaIDH was completely NADP(+)-dependent and no NAD(+)-linked activity was detectable. The K m values for NADP(+) were 32.24 and 71.71 μM with Mg(2+) and Mn(2+) as a sole divalent cation, and DL-isocitrate linked K m values were 32.56 μM (Mg(2+)) and 124.3 μM (Mn(2+)), respectively. As compared with Mn(2+), MaIDH showed about 2.5-times and 4-times higher affinities (1/K m) to NADP(+) and DL-isocitrate with Mg(2+). The optimum activity of MaIDH was found at pH 7.5, and its optimum temperature was 45 °C (Mn(2+)) and 50 °C (Mg(2+)). Heat-inactivation studies showed that heat treatment for 20 min at 45 °C caused a 50 % loss of enzyme activity. MaIDH was completely divalent cation dependent as other typical dimeric IDHs and Mn(2+) was its best activator. Our study is expected to give a better understanding of primary metabolic enzymes in M. aeruginosa. This would provide useful basic information for the research of controlling the blue-green algae blooms through biological techniques.
authors.affiliation: Key Laboratory of Molecular Evolution and Biodiversity and Institute of Molecular Biology and Biotechnology, College of Life Sciences, Anhui Normal University, No.1 Beijing East Road, Wuhu 241000, Anhui, People's Republic of China.
authors.forename: Ming-Ming
authors.initials: MM
authors.lastname: Jin
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.forename: Xue
authors.initials: X
authors.lastname: Li
authors.forename: Xiao-Yu
authors.initials: XY
authors.lastname: Zhao
authors.forename: Lei
authors.initials: L
authors.lastname: Xu
authors.forename: Ping
authors.initials: P
authors.lastname: Song
authors.forename: Guo-Ping
authors.initials: GP
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004734:Energy Metabolism; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007523:Isocitrates / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D046931:Microcystis / Q000201:enzymology*; D009249:NADP / Q000737:chemistry
chemical_list: D007523:Isocitrates; D007656:Ketoglutaric Acids; D009249:NADP; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)