Biophysical analysis of heat-induced structural maturation of glutamate dehydrogenase from a hyperthermophilic...

Tertiary structure of the recombinant glutamate dehydrogenase from Thermococcus kodakaraensis KOD1 (Tk-rGDH) converts into an intact form induced by the heat treatment. This phenomenon, heat-induced structural maturation, means that high temperature plays an important role in the proper folding and oligomerization of Tk-rGDH. In this work, we analyzed the heat-induced structural maturation of Tk-rGDH by differential scanning microcalorimetry (DSC), circular dichroism (CD), and activity measurements. In DSC measurements, the peak of adsorption of non-heated Tk-rGDH (nh-Tk-rGDH) was two times smaller than that of Tk-rGDH heated at 70 degrees C for 30 min (h-Tk-rGDH). The transition temperature (T(m)) of h-Tk-rGDH was 115 degrees C, which was about 3 degrees C higher than that of nh-Tk-rGDH. In the presence of 0.5 M NaCl, the nh-Tk-rGDH showed two peaks at 107 degrees C and 114 degrees C, while the h-Tk-rGDH showed a single peak at 115.7 degrees C. The heat-induced conformational change process was monitored by changes in CD intensity at 222 nm, and the result showed that heat-induced structural maturation is irreversible. The heat treatment at 70 degrees C showed the highest enhancement in activity, which was 15% larger than that of heat-treated Tk-rGDH at 40 degrees C. The results indicate that heat-induced structural maturation involves an irreversible process, transforming the non-heated form to the stable and active form.