The 2.9A resolution crystal structure of malate dehydrogenase from Archaeoglobus fulgidus: mechanisms of oligomerisation and thermal stabilisation.

The crystal structure of malate dehydrogenase from the hyperthermophilic archaeon Archeoglobus fulgidus, in complex with its cofactor NAD, was solved at 2.9A resolution. The crystal structure shows a compact homodimer with one coenzyme bound per subunit. The substrate binding site is occupied by a sulphate ion. In order to gain insight into adaptation mechanisms, which allow the protein to be stable and active at high temperatures, the 3D structure was compared to those of several thermostable and hyperthermostable homologues, and to halophilic malate dehydrogenase. The hyperthermostable A. fulgidus MalDH protein displays a reduction of the solvent-exposed surface, an optimised compact hydrophobic core, a high number of hydrogen bonds, and includes a large number of ion pairs at the protein surface. These features occur concomitantly with a reduced number of residues in the protein subunit, due to several deletions in loop regions. The loops are further stiffened by ion pair links with secondary structure elements. A. fulgidus malate dehydrogenase is the only dimeric protein known to date that belongs to the [LDH-like] MalDH family. All the other known members of this family are homo-tetramers. The crystal structures revealed that the association of the dimers to form tetramers is prevented by several deletions, taking place at the level of two loops that are known to be essential for the tetramerisation process within the LDH and [LDH-like] MalDH enzymes.

Crystallization and preliminary X-ray structure analysis of isocitrate dehydrogenase from two hyperthermophiles, Aeropyrum pernix and Thermotoga maritima.

Isocitrate dehydrogenase (IDH) catalyses the dehydrogenation and decarboxylation of isocitrate to alpha-ketoglutarate and CO(2) with NAD or NADP as cofactor. IDH from Aeropyrum pernix is the most thermostable IDH identified. Crystals of A. pernix IDH diffracted to 2.6 A with synchrotron radiation and belong to space group P4(3)2(1)2. IDH from Thermotoga maritima is the only IDH that has been characterized as homotetrameric and might be an evolutionary link between two different IDH subfamilies. T. maritima IDH crystals diffracted to 2.8 A with Cu Kalpha radiation and belong to space group P2(1)2(1)2(1). The structures will be helpful in the study of the factors responsible for thermostability and the evolutionary relationships of IDHs.

Identification of cofactor discrimination sites in NAD-isocitrate dehydrogenase from Pyrococcus furiosus.

The role of Asp-328 and Ile-329 as a cofactor discrimination site of the NAD-dependent isocitrate dehydrognase (NAD-IDH) from Pyrococcus furiosus has been verified by replacing these residues with Lys and Tyr, respectively, which are the corresponding residues in NADP-IDH from Escherichia coli. The Asp-328-Lys mutant showed dual coenzyme specificity, whereas introduction of the double mutation, Asp-328-Lys/Ile-329-Tyr shifted the cofactor preference from NAD to NADP. NADP-dependent P. furiosus IDH retained thermostability and thermoactivity compared with NAD-IDH.