Biochemical characterization of NADP+-dependent isocitrate dehydrogenase from Microcystis aeruginosa PCC7806
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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 Mg2+ and Mn2+ as a sole divalent cation, and DL-isocitrate linked K m values were 32.56 μM (Mg2+) and 124.3 μM (Mn2+), respectively. As compared with Mn2+, MaIDH showed about 2.5-times and 4-times higher affinities (1/K m) to NADP+ and dl-isocitrate with Mg2+. The optimum activity of MaIDH was found at pH 7.5, and its optimum temperature was 45 °C (Mn2+) and 50 °C (Mg2+). 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 Mn2+ 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.
KeywordsMicrocystis aeruginosa Isocitrate dehydrogenase Biochemical properties Coenzyme specificity
This research was supported by funds from the National High Technology Research and Development Program (“863” Program: 2012AA02A708), the National Natural Science Foundation of China (31170005; 30900243), Specialized Research Fund for the Doctoral Program of Higher Education of China (20113424110004), the Fund of State Key Laboratory of Genetics Resources and Evolution from Kunming Institute of Zoology (Chinese Academy of Sciences, CAS) (GREKF11-07), Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources and Program for Innovative Research Team in Anhui Normal University.
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