Elucidation of stability determinants of cold-adapted monomeric isocitrate dehydrogenase from a psychrophilic bacterium, Colwellia maris, by construction of chimeric enzymes

Seiya WatanabeYasuhiro Takada


To elucidate determinants of differences in thermostability between mesophilic and psychrophilic monomeric isocitrate dehydrogenases (IDHs) from Azotobacter vinelandii (AvIDH) and Colwellia maris (CmIDH), respectively, chimeric enzymes derived from the two IDHs were constructed based on the recently resolved three-dimensional structure of AvIDH, and several characteristics of the two wild-type and six chimeric IDHs were examined. These characteristics were then compared with those of dimeric IDH from Escherichia coli (EcIDH). All recombinant enzymes with a (His)(6)-tag attached to the N-terminal were overexpressed in the E. coli cells and purified by Ni(2+)-affinity chromatography. The catalytic activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) of the wild-type AvIDH and CmIDH were higher than those of EcIDH, implying that an improved catalytic rate more than compensates for the loss of a catalytic site in the former two IDHs due to monomerization. Analyses of the thermostability and kinetic parameters of the chimeric enzymes indicated that region 2, corresponding to domain II, and particularly region 3 located in the C-terminal part of domain I, are involved in the thermolability of CmIDH, and that the corresponding two...Continue Reading


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Related Concepts

Adaptation, Physiological
Cold Temperature
DNA, Bacterial
Enzyme Stability
Alkalescens-Dispar Group
Isocitrate Dehydrogenase-I
Protein Conformation
Chimeric Proteins, Recombinant
Azotobacter vinelandii

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