Insights into the molecular basis of thermal stability from the structure determination of Pyrococcus furiosus gluatamate dehydrogenase

Abstract

The structure determination of the glutamate dehydrogenase from the hyperthermophile Pyrococcus.fitriosus has been completed at 2.2 A resolution. The structure has been compared with the glutamate dehydrogenases from the mesophiles CIostridium symbiosum, Escherichia coli and Neurospora crassa. This comparison has revealed that the hyperthermophilic enzyme contains a striking series of networks of ion-pairs which are formed by regions of the protein which contain a high density of charged residues. Such regions are not found in the mesophilic enzymes and the number and extent of ion-pair formation is much more limited. The ion-pair networks are clustered at both inter domain and inter subunit interfaces and may well represent a major stabilising feature associated with the adaptation of enzymes to extreme temperatures.

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

“…This key fact emerged from the formation of two hydrogen bonds by two-counterpart arginine residues, and also by those of Ala141 residues belonging to each subunit of the dimer, which was found as the only possible prediction from our model. Although the T m of the Q144R mutant enzyme is nearly the same as that of Cs-GluDH, 36) the equivalent position of Arg144 in the Bs-GluDH Q144R mutant is Arg153 in Cs-GluDH, which, unlike the A-F dimer, makes intersubunit interaction by hydrogen bond formation between the A-D dimer (Arg153NH1-Ile183.O and Arg153NH2-Lys182.O). However, based on our model, we expect that in the inter-subunit interaction, Arg144 forms hydrogen bonds with its identical counterpart in the A-F dimer, or it might additionally form interactions in the A-D dimer like that of Cs-GluDH, which might be detectable by a more precise model.…”

“…The ion-pair networks are clustered at both inter-domain and inter-subunit interfaces, and might well represent a major stabilizing feature associated with the adaptation of enzymes to extreme temperatures. 36) In Pf-GluDH, the most dramatic ionpair cluster lies at the interface between the two-fold axis relating dimers (dimer AF-dimer CD) in this hexameric protein, and involves the linkage of 18 charged residues. 37) On the other hand, the number of inter-subunit ion-pairs in PiGluDH molecules is much smaller than those in PfGluDH.…”

Two Approaches to the Study of the Origin of Life

“…30°C and 40°C. For higher temperatures, special adjustments are found, as in the hyperthermophiles (Jaenicke 1996; Rice et al 1996). This constancy of temperature is conditional on the operation of a highly organised, dynamic system and is therefore basic to a systems approach to biogenesis.…”

“…In eukaryotes, doubling of the chromonemata enabled their meiotic reshuffling with another one from a genetically independent cell, which once more reduces the chance of mutations protracting considerably (Bernstein and Bernstein 1997). Similarly, in proteins of particularly hyperthermophile bacteria, ion pairs between glutamates and amino acids stabilise the molecule against effects of high temperatures (Jaenicke 1996; Rice et al 1996).…”

Extremely thermophilic microorganisms and their polymer-hidrolytic enzymes

“…The ion-pair networks are clustered at both inter domain and inter subunit interfaces. They may well represent a major stabilising feature associated with the adaptation of enzymes to extreme temperatures (39).…”