Structural studies of Burkholderia pseudomallei invasion protein D (BipD), 2,4'-dihydroxyacetophenone dioxygenase (DAD) and L-threonine dehydrogenase (TDH)

The determination of novel protein structures is essential in advancing our understanding of how they function within their molecular environments, therefore providing some of the information necessary to exploit them in ways that will benefit society. Three very different novel proteins have been studied by X-ray crystallography, and it is hoped that the information gained can be applied to real life challenges. Burkholderia pseudomallei invasion protein D (BipD) not only has the potential for misuse as a bioterrorist weapon, due to its air-borne infectivity, but also causes the disease melioidosis, which can kill within 48 hours. The structure of this novel protein was determined by MAD phasing methods to 2.1 A, and has been found to be highly homologous with IpaD from Shigella flexneri. This knowledge has enabled proposals to be made for a homopentameric ring structure of this protein and identified possible pH induced conformational changes which may have relevance in vivo. 2,4’-Dihydroxyacetophenone Dioxygenase (DAD) from Alcaligenes sp. is of interest for quite a different reason. Aromatic hydrocarbons, are especially damaging to the environment because they are not naturally degraded. The expression and purification protocols for DAD have now been refined and the native protein has been crystallised and diffraction data collected. However, because it is a novel structure, a selenomethionine derivative must also be crystallised and data collected; these optimisation trials are continuing. Structural knowledge about the enzymes involved in aromatic catabolic pathways will result in a better understanding of the whole system and therefore present opportunities for manipulation for bioremediation purposes. Knowledge of a protein’s structure can also help identify how specific properties are conferred, such as thermostability at extreme temperatures. L-Threonine Dehydrogenase from the hyperthermophilic archaeon Thermococcus kodakaraensis (TkTDH) is involved in the first of a two-step biochemical pathway conversion of threonine to glycine, by catalysing the NAD+ dependent metabolism of L-threonine to 2-amino-3-ketobutyrate (KBL). The structure of this enzyme has been determined to 2.4 A bound to its co-factor NAD+. It has been proposed that a structural and catalytic zinc are present in each monomer of this homotetramer as observed in the structurally similar alcohol dehydrogenases, but both were absent in this structure. The conserved residues postulated to co-ordinate these zinc ions were however in conformation that would allow metal ion coordination. Attempts have also been made to model the TDH tetramer with 2 dimers of KBL, as it is though that these enzymes form a complex and pass the intermediate substrate between them. This modelling is continuing. The structures of BipD and TkTDH have been solved and work is continuing on DAD This has made available a large amount of structural and functional information that would otherwise be inaccessible, contributing to our overall understanding of these macromolecules, as well as providing specific information that can be applied to particular challenges.EThOS - Electronic Theses Online ServiceGBUnited Kingdo