Identificaiton Of A NADP~+-Dependent Isocitrate Dehydrogenase From Microcystis Aeruginosa And Investigaiton Of The Molecular Evolution Mechanism For Monomeric Isocitrate Dehydrogenase

Isocitrate dehydrogenase （IDH） is a key enzyme in tricarboxylic acid andplays important roles in energy and biosynthesis metabolisms. This enzyme iswidely distributed in all three domains of life: Bacteria, Archaea and Eukarya.Based on the coenzyme specificity, IDH can be divided into NAD⁺-dependent IDH（EC1.1.1.41） and NADP⁺-dependent IDH （EC1.1.1.42）. According to the numberof subunits, IDHs primarily consist of two types, homodimeric IDHs andmonomeric IDHs. Structural comparison indicates that monomeric IDHs evolvedfrom the dimeric IDHs through domain fusion, gene duplication, insertions anddeletions.Microcystis aeruginosa is the key symptom of water eutrophication andproduces persistent microcystins. However, the information on the enzymesinvolved in the central metabolism is scarce. In this study, the gene encoding IDHof M. aeruginosa （MaIDH） was cloned and overexpressed in Escherichia coliRosetta （DE3）. The analysis of SDS-PAGE and gel filtration chromatographyrevealed that the active form of MaIDH was homodimeric and the molecular massof subunit was estimated to be52.6kDa. Kinetic studies indicated that MaIDH wascompletely NADP⁺-dependent. MaIDH activity was activated by Mg²⁺and Mn²⁺.The maximum activity of MaIDH was around45°C (Mn²⁺) and50°C (Mg²⁺),respectively. The optimal pH of MaIDH was7.5. Heat-inactivation studies showedthat incubation at45°C for20mins caused50%loss of enzyme activity. In thepresence of Mg²⁺and Mn²⁺, the K_mvalues of MaIDH for NADP+were32.24μMand71.71μM and the K_mvalues for DL-isocitrate were32.56μM and124.3μM,respectively. MaIDH performed about2.5-times and4-times higher affinities （1/K_m）to NADP+and DL-isocitrate with Mg²⁺than those with Mn²⁺, respectively.In order to investigate the evolutionary mechanism of monomeric IDHs, E.coli IDH （EcIDH） was engineered. EcIDH and MaIDH are in the same subfamilly, and the crystal structure of EcIDH has been resolved. After engineering, themonomeric EcIDH （MonoEcIDH） was constructed. The molecular weight ofMonoEcIDH was about62kDa. In the presence of Mg²⁺, the K_mvalues ofMonoEcIDH for NADP+and DL-isocitrate were34.79μM and33.62μM,respectively, indicating that MonoEcIDH has high affinities （1/K_m） to NADP+andDL-isocitrate, respectively. However, the catalytic efficiency （kcat/K_m） was onlyabout1/100of other monomeric IDHs. Further engineering works to improve thecatalytic efficiency of MonoEcIDH are in progress. Our study is expected to give abetter understanding of primary metabolic enzymes in M. aeruginosa, which wouldprovide useful basic information for the research of controlling the blue-green algaeblooms through biological techniques. The research of modification from E. colidimeric NADP-IDH to monomeric NADP-IDH provides useful experimentalevidences of the evolutionary relationship between monomeric IDH andhomodimeric IDH.The enzymatic characterization of M. aeruginosa IDH might be useful forcontrol the contamination of M. aeruginosa due to the critical roles of IDH in TCAcycle. E. coli dimeric NADP-IDH was engineered into monomeric NADP-IDH,which provides experimental evidence for the hypothesis that the monomeric IDHmight evolved from dimeric IDH.