Crystallization of the NAD(P)-dependent glutamate dehydrogenase from the hyperthermophilePyrococcus furiosus

Abstract

The NAD(P)-dependent glutamate dehydrogenase from Pyrococcus furiosus has been crystallized by the hanging-drop method of vapour diffusion using lithium sulfate as the precipitant. The crystals belong to the tetragonal system and are in space group P422~2 with unit-cell dimensions of a = b = 167.2, c = 172.9 A,. Consideration of the values of Vm and possible packing of the molecules within the cell suggest that the asymmetric unit contains a trimer. P. furiosus belongs to the family of Archaea and is one of the most thermostable organisms known, having an optimal growth temperature of 376 K. The glutamate dehydrogenase isolated from this organism has a half-life of 12 h at 373 K and, therefore, the determination of the structure of this enzyme will be important in advancing our understanding of how proteins are adapted to enable them to survive at such extreme temperatures.

The structure of Pyrococcus furiosus glutamate dehydrogenase reveals a key role for ion-pair networks in maintaining enzyme stability at extreme temperatures

“…The results of such an analysis have to be interpreted carefully, because crystal packing contacts can have a significant effect on the observed temperature factors. The Vm of the Pf enzyme is unusually high at 4.2 A 3 Da-l [22] and the crystal lattice is dominated by the presence of cylindrical cavities between the molecules, each 70 A long and 80 A in diameter, with only a small number of contacts between the molecules involving residues from domain II. In contrast, the Vm for Cs GluDH is 2.6 A 3 Da -l [23], which is close to the average observed for protein crystals [24] and there are a large number of lattice contacts involving atoms from both domains.…”

“…GluDH from P. furiosus was purified using the procedure described previously [22] and crystallized by the hanging drop method of vapour diffusion using lithium sulphate as the precipitant [22]. The crystals belong to space group P4 2 2 1 2; with cell dimensions a=b=167.2 A, c=173.0 A.…”

Crystallization and preliminary X-ray crystallographic studies on maltosyltransferase fromThermotoga maritima

“…If this is the case, then given a molecular weight of 73.7 kDa, the V m value of 4.0 A Ê 3 Da À1 corresponds to a rather high solvent content in the unit cell (about 66%; Matthews, 1968). However, this is not unusual for proteins isolated from hyperthermophiles (Yip et al, 1995;Tahirov et al, 1998). Our efforts are currently being directed towards a search for heavy-atom derivatives and the solution of the structure using multiple isomorphous replacement.…”

From hyperthermophiles to psychrophiles: the structural basis of temperature stability of the amino acid dehydrogenases

“…Analysis of the structure of hyperthermophilic proteins has highlighted a range of differences between these enzymes and their mesophilic counterparts, including increased numbers of ion pairs, reduction in the size of loops and in the number of cavities, reduced ratio of surface area to volume, introduction of additional proline residues, increased hydrophobic interaction at subunit interfaces, increased extent of secondary structure formation and truncated N-and C-termini [2][3][4][5][6][7][8][9]. The changes in stabilization energy required to adapt an enzyme to high-temperature operation are thought to be only of the order of a few hydrogen bonds, van der Waals contacts or stabilizing ion pairs [10].…”