Two Different Isocitrate Dehydrogenases from : Enzymology and Coenzyme-Evolutionary Implications.

PAO1, as an experimental model for Gram-negative bacteria, harbors two NADP-dependent isocitrate dehydrogenases (NADP-IDHs) that were evolved from its ancient counterpart NAD-IDHs. For a better understanding of PaIDH1 and PaIDH2, we cloned the genes, overexpressed them in and purified them to homogeneity. PaIDH1 displayed higher affinity to NADP and isocitrate, with lower Km values when compared to PaIDH2. Moreover, PaIDH1 possessed higher temperature tolerance (50 °C) and wider pH range tolerance (7.2-8.5) and could be phosphorylated. After treatment with the bifunctional PaIDH kinase/phosphatase (PaIDH K/P), PaIDH1 lost 80% of its enzymatic activity in one hour due to the phosphorylation of Ser115. Small-molecule compounds like glyoxylic acid and oxaloacetate can effectively inhibit the activity of PaIDHs. The mutant PaIDH1-D346I347A353K393 exhibited enhanced affinity for NAD while it lost activity towards NADP, and the Km value (7770.67 μM) of the mutant PaIDH2-L589 I600 for NADP was higher than that observed for NAD (5824.33 μM), indicating a shift in coenzyme specificity from NADP to NAD for both PaIDHs. The experiments demonstrated that the mutation did not alter the oligomeric state of either protein. This study provides a foundation for the elucidation of the evolution and function of two NADP-IDHs in the pathogenic bacterium .