This application offers a thorough structural dissection of the hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) enzyme from Toxoplasma gondii, a protozoan parasite that wreaks havoc in individuals with AIDS. The goal of this proposal is to characterize the HGXPRT protein in atomic detail employing a combination of X-ray crystallography, computational chemistry, biochemistry, and molecular biology. The first specific aim of this grant will be to improve the resolution of the structure of the HGXPRT apoenzyme to its limiting resolution, i.e., 2.0 A.
Specific Aim II will involve an analysis of product-enzyme complexes, structures which are essential for a complete mechanistic understanding of the protein and its substrate specificity.
Specific Aim III will be to evaluate the structure of the T. gondii HGXPRT by site-directed muta-genesis of key amino acid residues that are predicted to be essential for catalysis or to govern substrate specificity. The penultimate specific aim will be to initiate a crystallographic analysis of the T. gondii HGXPRT complexed to substrate and/or substrate analogs, information that should provide direct insight into substrate recognition and binding by the protein. The last specific aim will be to implement computational screens of 3-D small molecule structural databases with our apoenzyme structure, and ultimately improved structures, to discover novel compounds that target the active site of the enzyme. These compounds will be evaluated biochemically on the enzyme in vitro and in vivo, and the crystal structures of the T. gondii HGXPRT complexed with the most promising lead compounds will be determined.
| Schumacher, M A; Scott, D M; Mathews, I I et al. (2000) Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding. J Mol Biol 298:875-93 |
| Schumacher, M A; Scott, D M; Mathews, I I et al. (2000) Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding. J Mol Biol 296:549-67 |
| Schumacher, M A; Carter, D; Scott, D M et al. (1998) Crystal structures of Toxoplasma gondii uracil phosphoribosyltransferase reveal the atomic basis of pyrimidine discrimination and prodrug binding. EMBO J 17:3219-32 |
