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.

Physiological Regulation of Isocitrate Dehydrogenase and the Role of 2-Oxoglutarate in Prochlorococcus sp. Strain PCC 9511

“…The samples without added manganese showed a 25% of the control activity (complete mixture), while those where manganese was replaced by magnesium had roughly half of the control activity, indicating a preference of ICDH for the manganese ion. Similar results have been observed in other bacteria, including cyanobacteria [36], [38], [39], [53], [54]. In a series of assays at different temperatures, we observed that ICDH activity increased continuously from 30 to 55°C, but at the same time it became less stable: preincubation of the cell extracts at different temperatures showed that the activity decreased sharply above 35°C.…”

“…9.0–9.5. This value is close to those described for other NADP-ICDH [35]–[38], [55]–[57], with the exception of M. aeruginosa , which had an optimum pH of 7.5 [39], although it has to be taken into account that this was a recombinant enzyme.…”

“…In a series of assays at different temperatures, we observed that ICDH activity increased continuously from 30 to 55°C, but at the same time it became less stable: preincubation of the cell extracts at different temperatures showed that the activity decreased sharply above 35°C. Similar results have been described in M. aeruginosa PCC 7806 [39]. Therefore, the standard assay temperature was set up at 40°C.…”

“…PCC 7120 [37], Phormidium laminosum [38] and Microcystis aeruginosa sp. PCC 7806 [39]), the latter expressed in E. coli , being composed of two identical subunits of 55 kDa. Besides, the gene icd , encoding ICDH, has been cloned from Synechocystis sp.…”

“…PCC 6803 [40], Anabaena sp. PCC 7120 [37], and Microcystis aeruginosa PCC 7806 [39], and it is found in all cyanobacterial genomes thus far available (including all the Prochlorococcus and Synechococcus strains). This fact, together with the impossibility to segregate icd mutants observed in Synechocystis sp.…”