Molecular cloning, purification, and biochemical characterization of Recombinant Isocitrate dehydrogenase from Streptomyces coelicolor M-145

Abstract

Isocitrate dehydrogenase is a key enzyme in carbon metabolism. In this study we demonstrated that SCO7000 of Streptomyces coelicolor M-145 codes for the isocitrate dehydrogenase. Recombinant enzyme expressed in Escherichia coli had a specific activity of 25.3 μmoles/mg/min using NADP+ and Mn2+ as a cofactor, 40-times higher than that obtained in cellfree extract. Pure IDH showed a single band with an apparent Mr of 84 KDa in SDS-PAGE, which was also recognized as His-tag protein in the Western blot. Unexpectedly, in ND-PAGE conditions showed a predominant band of ∼168 KDa that corresponded to the dimeric form of ScIDH. Also, zymogram assay and analytical gel filtration reveal that dimer was the active form. Kinetic parameters were 1.38, 0.11, and 0.109 mM for isocitrate, NADP, and Mn2+, respectively. ATP, ADP, AMP, and their mixtures were the main ScIDH activity inhibitors. Zn2+, Mg2+, Ca2+, and Cu+ had inhibitory effect on enzyme activity.