Structure determination of the glutamate dehydrogenase from the hyperthermophile Thermococcus litoralis and its comparison with that from Pyrococcus furiosus

Glutamate dehydrogenase catalyses the oxidative deamination of glutamate to 2-oxoglutarate with concomitant reduction of NAD(P)(+), and has been shown to be widely distributed in nature across species ranging from psychrophil es to hyperthermophiles. Extensive characterisation of this enzyme isolated from hyperthermophilic organisms has led to its adoption as a model system for analysing the determinants of thermal stability. The crystal structure of the extremely thermostable glutamate dehydrogenase from Thermococcus li toralis has been determined at 2.5 Angstrom resolution, and has been compar ed to that from the hyperthermophile Pyrococcus furiosus. The two enzymes a re 87 % identical in sequence, yet differ 16-fold in their half-lives at 10 4 degrees C. This is the first reported comparative analysis of the structu res of a multisubunit enzyme from two closely related yet distinct hyperthe rmophilies. The less stable T. litoralis enzyme has a decreased number of i on pair interactions; modified patterns of hydrogen bonding resulting from isosteric sequence changes; substitutions that decrease packing efficiency; and substitutions which give rise to subtle but distinct shifts in both ma in-chain and side-chain elements of the structure. This analysis provides a rational basis to test ideas on the factors that confer thermal stability in proteins through a combination of mutagenesis, calorimetry, and structur al studies. (C) 1999 Academic Press.