Heat effect on the structure and activity of the recombinant glutamate dehydrogenase from a hyperthermophilic archaeon Pyrococcus horikoshii

Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290±8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90 °C, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100 °C was 4 h). The enzyme shows strict specificity for 2-oxoglutarate and l-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. Km values of the recombinant enzyme are comparable for both substrates: 0.2 mM for l-glutamate and 0.53 mM for 2-oxoglutarate. The enzyme was activated by heating at 80 °C for 1 h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent.