EXCHANGE OF DOMAINS OF GLUTAMATE-DEHYDROGENASE FROM THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-FURIOSUS AND THE MESOPHILIC BACTERIUM CLOSTRIDIUM-DIFFICILE - EFFECTS ON CATALYSIS, THERMOACTIVITY AND STABILITY

The glutamate dehydrogenase gene from the hyperthermophilic archaeon P yrococcus furiosus has been functionally expressed in Escherichia coli under the control of the lambda, P-L promoter. The P.furiosus glutama te dehydrogenase amounted to 20% of the total E.coli cell protein, and the vast majority consisted of hexamers, Following activation by heat treatment, an enzyme could be purified from E,coli that was indisting uishable from the glutamate dehydrogenase purified from P.furiosus. Hy brid genes, that consisted of the coding regions for the homologous gl utamate dehydrogenases from P.furiosus and the mesophilic bacterium Cl ostridium difficile, were constructed and successfully expressed in E, coli, One of the resulting hybrid proteins, containing the glutamate b inding domain of the C,difficile enzyme and the cofactor binding domai n of the P.furiosus enzyme, did not show a detectable activity, In con trast, the complementary hybrid containing the P.furiosus glutamate an d the C.difficile cofactor binding domain was a catalytically active h examer that showed a reduced substrate affinity but maintained efficie nt cofactor binding with the specificity found in the Clostridium symb iosum enzyme, Compared with the C,difficile glutamate dehydrogenase, t he archaeal-bacterial hybrid is slightly more thermoactive, less therm ostable but much more stable towards guanidinium chloride-induced inac tivation and denaturation.