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.

The discovery of hyperthermophilic microorganisms and the analysis of hyperthermostable enzymes has established the fact that multisubunit enzymes can survive for prolonged periods at temperatures above 100°C. We have carried out homology-based modeling and direct structure comparison on the hexameric glutamate dehydrogenases from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis whose optimal growth temperatures are 100°C and 88°C, respectively, to determine key stabilizing features. These enzymes, which are 87% homologous, differ 16-fold in thermal stability at 104°C. We observed that an intersubunit ion-pair network was substantially reduced in the less stable enzyme from T. litoralis, and two residues were then altered to restore these interactions. The single mutations both had adverse effects on the thermostability of the protein. However, with both mutations in place, we observed a fourfold improvement of stability at 104°C over the wild-type enzyme. The catalytic properties of the enzymes were unaffected by the mutations. These results suggest that extensive ion-pair networks may provide a general strategy for manipulating enzyme thermostability of multisubunit enzymes. However, this study emphasizes the importance of the exact local environment of a residue in determining its effects on stability.

The structure of glucose dehydrogenase from the extreme halophile Haloferax mediterranei has been solved at 1.6-Å resolution under crystallization conditions which closely mimic the ''in vivo'' intracellular environment. The decoration of the enzyme's surface with acidic residues is only partially neutralized by bound potassium counterions, which also appear to play a role in substrate binding. The surface shows the expected reduction in hydrophobic character, surprisingly not from changes associated with the loss of exposed hydrophobic residues but rather arising from a loss of lysines consistent with the genome wide-reduction of this residue in extreme halophiles. The structure reveals a highly ordered, multilayered solvation shell that can be seen to be organized into one dominant network covering much of the exposed surface accessible area to an extent not seen in almost any other protein structure solved. This finding is consistent with the requirement of the enzyme to form a protective shell in a dehydrating environment.Archaea ͉ x-ray structure ͉ water structure ͉ hydrophobic surface ͉ surface lysines

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The recent structure determination of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus and the comparison of this structure with its counterparts from the mesophiles Clostridium symbiosum and Escherichia coli has highlighted the formation of extended networks of ion-pairs as a possible explanation for the superior thermal stability of the hyperthermostable enzyme. In the light of this, we have carried out a homology-based modelling study using sequences of a range of glutamate dehydrogenases drawn from species which span a wide spectrum of optimal growth temperatures. We have attempted to analyse the extent of the formation of ion-pair networks in these different enzymes and tried to correlate this with the observed thermal stability. The results of this analysis indicate that the ion-pair networks become more fragmented as the temperature stability of the enzyme decreases and are consistent with a role for the involvement of such networks in the adaptation of enzymes to extreme temperatures.Keywords : glutamate dehydrogenase; thermal stability; homology modeling; ion-pair network; Archaea.A proper understanding of the molecular basis of thermal ophiles has permitted direct structural comparisons, homologybased modelling studies and site-directed mutagenesis to be unstability in proteins could have important consequences for their application in a range of biotechnological processes. For exam-dertaken, combining sequence data on extremophilic enzymes with structural data from their mesophilic counterparts. Howple, the availability of enzymes with the appropriate specificity and capable of surviving for long periods at extreme temper-ever, to date, these analyses have not produced a consistent picture on the origins of thermal stability and suggestions put foratures could lead to the creation of novel applications of enzyme-based technology in industries such as those involved in ward to explain enhanced stability properties of proteins have included decreased flexibility of the protein [4Ϫ5] arising from the processing of paper, pulp or fibres [1Ϫ2]. The opportunities afforded by such applications have generated significant interest changes in interactions such as the increase in the number of ion-pairs [6Ϫ9], an increase in packing density [10], a decrease in the field and a large number of studies have been undertaken to unravel the molecular mechanisms involved in generating in the degree of cavity formation [11Ϫ12], a decrease in the sizes of loops linking secondary-structure elements [11], an thermostable enzymes. Recently, increasing attention has been focused on proteins from Archaea, a phylogenetically distinct increase in alanine residues located at the termini of surface A helices [13], a reduction in exposed surface area [14], an evolutionary kingdom that includes many extremophilic microorganisms and whose study has expanded our horizons on the increase in proline content [15Ϫ16], an increase in the number of disulphide bonds [17] and an increase in hydrophobic interaclimits of b...

The family of glutamate dehydrogenases include a group of hexameric oligomers with a subunit Mr of around 50000, which are closely related in amino acid sequence and a smaller group of tetrameric oligomers based on a much larger subunit with Mr 115000. Sequence comparisons have indicated a low level of similarity between the C‐terminal portion of the tetrameric enzymes and a substantial region of the polypeptide chain for the more widespread hexameric glutamate dehydrogenases. In the light of the solution of the three dimensional structure of the hexameric NAD+‐linked glutamate dehydrogenase from Clostridium symbiosum, we have undertaken a detailed examination of the alignment of the sequence for the C‐terminal domain of the tetrameric Neurospora crassa glutamate dehydrogenase against the sequence and the molecular structure of that from C. symbiosum. This analysis reveals that the residues conserved between these two families are clustered in the three‐dimensional structure and points to a remarkably similar layout of the glutamate‐binding site and the active‐site pocket, though with some differences in the mode of recognition of the nucleotide cofactor.

In the light of the solution of the three-dimensional structure of the NAD(+)-linked glutamate dehydrogenase from the mesophile Clostridium symbiosum, we have undertaken a detailed examination of the alignment of the sequences for the thermophilic glutamate dehydrogenases from Thermococcus litoralis and Pyrococcus furiosus against the sequence and the molecular structure of the glutamate dehydrogenase from C. symbiosum, to provide insights into the molecular basis of their thermostability. This homology-based modelling is simplified by the relatively small number of amino acid substitutions between the two thermophilic glutamate dehydrogenase sequences. The most frequent amino acid exchanges involve substitutions which increase the hydrophobicity and sidechain branching in the more thermostable enzyme; particularly common is the substitution of valine to isoleucine. Examination of the sequence differences suggests that enhanced packing within the buried core of the protein plays an important role in maintaining stability at extreme temperatures. One hot spot for the accumulation of exchanges lies close to a region of the molecule involved in its conformational flexibility and these changes may modulate the dynamics of this enzyme and thereby contribute to increased stability.