Insights into Thermal Stability from a Comparison of the Glutamate Dehydrogenases from Pyrococcus furiosus and Thermococcus litoralis

Abstract

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 Thennococcus 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 homologybased 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.

Rigidity versus flexibility: the dilemma of understanding protein thermal stability

“…This difference correlates with the average increase of the void volume (the volume of the interatomic space in the molecular interior where no other atoms can be inserted) of proteins from thermophiles suggesting that these molecules may have lower packing density. Opposite observa-tions are also reported: proteins from thermo extremophiles are characterized by a somewhat more compact hydrophobic core [47,[62][63][64]. This contradiction may have two sources.…”

Heat and SDS insensitive NDK dimers are largely stabilised by hydrophobic interaction to form functional hexamer in Mycobacterium smegmatis.

“…In general, thermophilic proteins/enzymes have a higher content of hydrophobic amino acids than that in the proteins of mesophiles, and these residues can increase the rigidity and hydrophobicity of proteins (Chakravarty & Varadarajan, 2000), thereby increasing their structural and functional integrity at high temperatures. Higher number of hydrophobic groups in thermophilic enzymes destabilises the unfolded forms gives more rigidity with increases in the temperature (Ikai et al, 1980;Britton et al, 1995). Reports from various research groups show that hydrophobic core and aliphatic side chains could also be responsible for protein stability at high temperatures (Argos et al, 1977;Lindsay, 1995;Haney et al, 1991;Vieille et al, 2001;Fütter et al, 2004;Irimia et al, 2004).…”