Eisatz
Abstract
The project will focus on two different enzymes: Pyranose 2-oxidase (P2O) anddUTPases. P2O is envisioned (and there are already some patents about this topic, Lit.) as a biocatalyst in carbohydrate transformation resulting in the production of prebiotic sugars, e.g. D-tagatose or lactulose. P2O is a homotetramer with an interesting structure. In a first step two subunits form a very stabile dimer. In a second step two of these dimers form the tetrameric, active form of the enzyme. From the scientific point of view analyzing of mutations on the contact surface of the subunits will allow a deeper insight in the mechanism of protein interaction. From the applied point of view thermostable mutants of P2O could be used at higher temperature, e.g. 50°C instead of 30°C, for transformation, reducing the risk of microbial infections, which is especially important in food industry. One aim of this project will be the construction of P2O variants containing mutations in the subunit surface and analyze their thermostability in detail to (i) gain new insights in the mechanism of protein unfolding and (ii) to perform conversion reactions at higher temperature without the fear of microbiological contaminations. The enzyme family of dUTPases is responsible for preventing incorporation of dUMP moieties into DNA. The enzyme is ubiquitous and essential – its lack leads to accumulation of uracil in DNA that triggers thymine-less cell death (an apoptotic pathway) (11). The enzyme is a novel and promising target for development of antimicorbial chemotherapeutics against bacterial, fungal, or viral infections and especially against tuberculosis and malaria (12,13). In addition, the human dUTPase is an important survival factor for multiresistant cancer cell lines – therefore its targeting offers design of novel anti-cancer drugs, as well (14,15). The 3D structure of the enzyme has been recently determined in our laboratory for several dUTPases (4,5,7,8). Based on the structures and on functional experiments in solution, we identified several factors involved in folding and specificity of dUTPases, but further key components need to be determined to provide a detailed understanding of structure and function of these enzymes. Formation of a unique homotrimeric arrangement is indispensable for function of dUTPases. The aims of this proposal is to investigate which interactions are crucial in creating the trimer and which contribute mostly to its stability. These results will be important to aid design of potential small molecular ligands that may efficiently interfere with dUTPase action and may then be proposed as lead molecules for inhibitor design.
keywords enzymes pyranose 2-oxidase dUTPase thermostability directed evolution
Publikationen
Project staff
Oliver Spadiut
Assoc. Prof. Dipl.-Ing. Dr.nat.techn. Oliver Spadiut
Project Staff
01.06.2008 - 31.05.2009
BOKU partners
External partners
Hungarian Academyof Sciences
Beata Vertessy
partner