Comparison of the structures of these two enzymes has revealed one major difference: the structure of the hyperthermophilic enzyme contains a striking series of ion-pair networks on the surface of the protein subunits and buried at both interdomain and intersubunit interfaces. We propose that the formation of such extended networks may represent a major stabilizing feature associated with the adaptation of enzymes to extreme temperatures.

No abstract

The hyperthermophilic archaebacterium Pyrococcus furiosus contains high levels of NAD(P)-dependent glutamate dehydrogenase activity. The enzyme could be involved in the first step of nitrogen metabolism, catalyzing the conversion of 2-oxoglutarate and ammonia to glutamate. The enzyme, purified to homogeneity, is a hexamer of 290 kDa (subunit mass 48 kDa). Isoelectric-focusing analysis of the purified enzyme showed a pl of 4.5. The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate but utilizes both NADH and NADPH as cofactors. The purified enzyme reveals an outstanding thermal stability (the half-life for thermal inactivation at 100°C was 12 h), totally independent of enzyme concentration.P. furiosus glutamate dehydrogenase represents 20% of the total protein; this elevated concentration raises questions about the roles of this enzyme in the metabolism of P..furiosus.Recently we reported the purification of an NAD(P)-dependent glutamate dehydrogenase from the thermophilic archaebacterium Sulfolobus so@ztaricus [l, 21. The enzyme of this microorganism is probably involved in the first step of ammonia assimilation by the following reaction: glutamate + NAD(P)+ + H 2 0 + 2-oxoglutarate + NH,' + NAD(P)H + H + .The glutamate dehydrogenase from S . solfutaricus presents some interesting properties; it has a double coenzyme specificity, is strictly specific for L-glutamate and 2-oxoglutarate and its thermal stability is a function of the enzyme concentration [2]. This latter property is in good agreement with the observed self-associating behaviour of the purified enzyme.Among archaebacterial glutamate dehydrogenases, the only data available for comparison are restricted to the halophilic phenotype [3]. Therefore, it is difficult to extrapolate from our observations on S. solfituricus glutamate dehydrogenase to obtain general rules for thermal adaptation in this class of enzymes and general knowledge regarding nitrogen metabolism in archaebacteria.To provide more information about the enzymological properties of archaebacterial glutamate dehydrogenases and to extend our investigations towards more thermostable forms of this enzyme, we attampted to purify glutamate dehydrogenase from the so-called 'hyperthermophiles'. These remarkable microorganisms, recently discovered, grow optimally at temperatures near 100 "C [4]. They all are archaebacteria and most of them are strict anaerobes and depend on the reduction of elemental sulfur for growth. So far, hyperthermophiles -___ have been classified into three distinct genera, Pyrodiictium, Pyrobaculurn and Pyrococcus. As a source of hyperthermophilic glutamate dehydrogenase we used Pyrococcus furiosus [5], one of the most interesting members of this last genus because of its relatively high cell yield and rapid growth rate. The microorganism grows optimally at 10O'C by a fermentative-type metabolism and is a strict heterotroph which utilizes both simple and complex carbohydrates and produces only H2 and C 0 2 as detectable products. P. furiosus reduces eleme...

Domain II (residues 189-338, M(r) = 16 222) of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was used as a model system to study reversible unfolding thermodynamics of this hyperthermostable enzyme. The protein was produced in large quantities in E.COLI: using a T7 expression system. It was shown that the recombinant domain is monomeric in solution and that it comprises secondary structural elements similar to those observed in the crystal structure of the hexameric enzyme. The recombinant domain is thermostable and undergoes reversible and cooperative thermal unfolding in the pH range 5.90-8.00 with melting temperatures between 75.1 and 68.0 degrees C. Thermal unfolding of the protein was studied using differential scanning calorimetry and circular dichroism spectroscopy. Both methods yielded comparable values. The analysis revealed an unfolding enthalpy at 70 degrees C of 70.2 +/- 4.0 kcal/mol and a DeltaC(p) value of 1.4 +/- 0.3 kcal/mol K. Chemical unfolding of the recombinant domain resulted in m values of 3.36 +/- 0.10 kcal/mol M for unfolding in guanidinium chloride and 1.46 +/- 0.04 kcal/mol M in urea. The thermodynamic parameters for thermal and chemical unfolding equilibria indicate that domain II from T.MARITIMA: glutamate dehydrogenase is a thermostable protein with a DeltaG(max) of 3.70 kcal/mol. However, the thermal and chemical stabilities of the domain are lower than those of the hexameric protein, indicating that interdomain interactions must play a significant role in the stabilization of T. MARITIMA: domain II glutamate dehydrogenase.

Background: ␤-Amyloid aggregates are at the basis of Alzheimer disease development. Short synthetic peptides are seen to inhibit polymerization. Results: Various synthetic peptides have been studied by MD simulations and tested experimentally. Conclusion:Combined results indicate Ac-LPFFN-NH 2 as an effective lead compound able to slow down A␤ 1-40 aggregation. Significance: Designing potential A␤ aggregation inhibitors will help fight Alzheimer disease.