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.

Hemolysin E (HlyE) is a novel pore-forming toxin of Escherichia coli, Salmonella typhi, and Shigella flexneri. Here we report the X-ray crystal structure of the water-soluble form of E. coli HlyE at 2.0 A resolution and the visualization of the lipid-associated form of the toxin in projection at low resolution by electron microscopy. The crystal structure reveals HlyE to be the first member of a new family of toxin structures, consisting of an elaborated helical bundle some 100 A long. The electron micrographs show how HlyE oligomerizes in the presence of lipid to form transmembrane pores. Taken together, the data from these two structural techniques allow us to propose a simple model for the structure of the pore and for membrane interaction.

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Comparison of the structure of leucine dehydrogenase with a hexameric glutamate dehydrogenase has shown that these two enzymes share a related fold and possess a similar catalytic chemistry. A mechanism for the basis of the differential amino acid specificity between these enzymes involves point mutations in the amino acid side-chain specificity pocket and subtle changes in the shape of this pocket caused by the differences in quaternary structure.

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...