Insights into the molecular basis of thermal stability from the analysis of ion‐pair networks in the Glutamate Dehydrogenase family

Abstract

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...

Molecular Properties and Enhancement of Thermostability by Random Mutagenesis of Glutamate Dehydrogenase fromBacillus subtilis

“…Although much information has been gained about the crystal structures of GluDHs from mesophilic and thermophilic microorganisms 26,27) and an account of the high thermostability of thermophilic GluDH has been given, [27][28][29] relatively few successful studies have been devoted to characterizing the thermostability of GluDHs from mesophilic bacteria. The unstable character of GluDH in Bacillus species was reported in the past decade.…”

Adaptive role of increased frequency of polypurine tracts in mRNA sequences of thermophilic prokaryotes

“…These factors include (i) a higher number of salt bridges (9)(10)(11)(12)(13)(14) [in this respect, it was suggested that not only a larger number of charged residues but also additional salt bridges around a particular bridge enhance the stability of the bridge (15,16)], (ii) additional hydrogen bonds (17)(18)(19)(20)(21), (iii) shorter loop regions (12,22), (iv) increasing intramolecular hydrophobic packing (11), and (v) the ␣-helical content of the proteins (23). These structural characteristics might result from different proportions of specific amino acids in the sequences of the thermophile proteins.…”

Crystal structure of a thermophilic alcohol dehydrogenase substrate complex suggests determinants of substrate specificity and thermostability

“…41 In this family the thermal stability correlates well with the charge density on the protein. 42 The most thermostable member of this family, the Pyrococcus furiosis enzyme with a melting temperature of 113°C, has a vast number of salt bridges, including ion-pair networks that involve up to 18 residues. 43 The existence of a strong correlation between the number of ion pairs and enzyme thermostability has also been corroborated by engineering extra salt bridges into the less thermostable glutamate dehydrogenase from Thermatoga maritima.…”