STRUCTURAL MECHANISM OF THE T GONDII UPRT, A TARGET FOR STRUCTURAL-BASED DRUG D

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The major research focus of the Brennan Laboratory is to understand the relationship between the 3-D structures of proteins and their biochemical and biological functions. These approaches are being used to study transcription regulation, protein-nucleic acid interaction, multidrug recognition and binding and the mechanisms of purine and pyrimidine salvage in protozoa. Our interest is also directed in structure-based drug design. One protein of interest is the uracil phosphoribosyltransferase (UPRT) from the opportunistic parasitic protozoan, Toxoplasma gondii, which catalyzes the transfer of uracil to D-phosphoribosyl pyrophosphate (PRPP) releasing pyrophosphate and creating the nucleotide monophosphate UMP. The salvaged UMP can then be used in a number of metabolic pathways. This enzyme presents a very good target to exploit in our attempts to discover new drugs against this opportunistic parasite. The substrate recognition and catalytic mechanisms of UPRT must be understood fully. Previous studies from our lab have set the groundwork for the complete structural and mechanistic characterization of UPRT and we are carrying out the next key set of studies. Specifically, we are working on the refinement of the ultra high resolution (1.05 ¿¿resolution) structure of apo UPRT and the high resolution structures of a number of key binary and ternary complexes. In parallel, we are carrying out a thorough kinetic evaluation of her wild type protein and a number of site directed mutants. Finally, this information starts to develop the structural parameters that will guide our future drug design attempts.