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.

The multi-layered structure of Dps with a novel di-nuclear ferroxidase center.

The crystallization of cellular components represents a unique survival strategy for bacterial cells under stressed conditions. A highly ordered, layered structure is often formed in such a process, which may involve one or more than one type of bio-macromolecules. The main advantage of biocrystallization has been attributed to the fact that it is a physical process and thus is independent of energy consumption. Dps is a protein that crystallizes to form a multi-layered structure in starved cells in order to protect DNA against oxidative damage and other detrimental factors. The multi-layered crystal structure of a Dps protein from Bacillus brevis has been revealed for the first time at atomic resolution in the absence of DNA. Inspection of the structure provides the first direct evidence for the existence of a di-nuclear ferroxidase center, which possesses unique features among all the di-iron proteins identified so far. It constitutes the structural basis for the ferroxidase activity of Dps in the crystalline state as well as in solution. This finding proves that the enzymatic process of detoxification of metal ions, which may cause severe oxidative damage to DNA, is the other important aspect of the defense mechanism performed by Dps. In the multi-layered structure, Dps dodecamers are organized in a highly ordered manner. They adopt the classic form of hexagonal packing in each layer of the structure. Such arrangement results in reinforced structural features that would facilitate the attraction and absorption of metal ions from the environment. The highly ordered layered structure may provide an ideal basis for the accommodation of DNA between the layers so that it can be isolated and protected from harmful factors under stress conditions.

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.

Human spermatid-specific thioredoxin-1 (Sptrx-1) is a two-domain protein with oxidizing activity.

Spermatid-specific thioredoxin-1 (Sptrx-1) is the first member of the thioredoxin family of proteins with a tissue-specific expression pattern, found exclusively in the tail of elongating spermatids and spermatozoa. We describe here further biochemical characterization of human Sptrx-1 protein structure and enzymatic activity. In gel filtration chromatography human Sptrx-1 eluates as a 400 kDa protein consistent with either an oligomeric form, not maintained by intermolecular disulfide bonding, and/or a highly asymmetrical structure. Analysis of circular dichroism spectra of fragments 1-360 and 361-469 and comparison to spectra of full-length Sptrx-1 supports a two-domain organization with a largely unstructured N-terminal domain and a folded thioredoxin-like C-terminal domain. Functionally, Sptrx-1 behaves as an oxidant in vitro when using selenite, but not oxidized glutathione, as electron acceptor. This oxidizing enzymatic activity suggests that Sptrx-1 might govern the stabilization (by disulfide cross-linking) of the different structures in the developing tail of spermatids and spermatozoa.