Contribution of Three Different Regions of Isocitrate Dehydrogenases from Psychrophilic and Psychrotolerant Bacteria to Their Thermal Properties.

Monomeric isocitrate dehydrogenases of a psychrophilic bacterium, Colwellia maris, and a psychrotolerant bacterium, Pseudomonas psychrophila, (CmIDH and PpIDH) are cold-adapted and mesophilic, respectively. On the other hand, previous studies revealed that the monomeric IDH of Azotobacter vinelandii (AvIDH) is also mesophilic and the regions 2 and 3 among three regions of this enzyme are involved in the thermal properties. Therefore, to examine whether the region(s) responsible for the mesophilic properties are common between PpIDH and AvIDH, the genes of chimeric IDHs exchanging three regions of PpIDH and CmIDH in various combinations were constructed and overexpressed as His-tagged recombinant proteins in the Escherichia coli cells, and the chimeric and wild-type PpIDH and CmIDH were purified with Ni-chelating affinity column chromatography. The swapping chimeras of the regions 2 or 3 in PpIDH and CmIDH showed lower and higher optimum temperatures for activities and their thermostabilities than the wild-type ones, respectively. On the other hand, the exchange of the respective region 1 hardly influenced these properties of the two IDHs. Therefore, the regions 2 and 3 of the two IDHs were confirmed to be involved in their thermal properties. These results were coincident with those of the previous study on chimeric IDHs between AvIDH and CmIDH, indicating that the common regions of AvIDH and PpIDH are responsible for their mesophilic properties and the amino acid residues involved in their thermal properties are present in the regions 2 and 3.