Amalgamating tools of molecular biology, genetics, biochemistry, structural biology, and immunochemistry, this proposal offers a thorough interdisciplinary analysis of three key enzymes of the purine salvage pathway of Leishmania. These enzymes are hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenine phosphoribosyltransferase (APRT), and xabthine phosphoribosyltransferase (XPRT). As protozoan parasites are auxotrophic for purines, HGPRT, APRT, and XPRT provide an important, if not vital, nutritional function for the parasite, and HGPRT initiates the intracellular metabolism of allopurinol, a lead compound that has shown therapeutic efficacy in both leishmaniasis and Chagas disease. The proposed investigations constitute a logical step in the validation of these enzymes as potential therapeutic targets and in the implementation of a rational, structure-based strategy of drug discovery, and ultimately drug design, for the treatment and prophylaxis of leishmaniasis and other diseases of parasitic origin. The first objective of this application is to determine the contributions of HGPRT, APRT, XPRT, and adenosine kinase (AK) to purine salvage in L. donovani promastigotes by phenotypic characterization of deltaxprt mutants that will be constructed by targeted gene replacement in wild type, deltahgprt, deltaaprt, deltahgprt/deltaaprt, deltahgprt/ak, deltaaprt/ak, and deltahgprt/deltaaprt/ak genetic backgrounds. Whether HGRPT, APRT, or XPRT function is essential for infectivity or virulence will be tested by generating null mutants in the infective M379 L. mexicana strain. The second specific aim entails a detailed biochemical and structural characterization of the HGRPT, APRT, and XPRT proteins. The first component of Specific Aim II consists of an evaluation of the HGPRT and XPRT molecular models and the APRT structure by site-directed mutagenesis of key amino acids that are conjectured to be involved in catalysis or substrate binding and biochemical characterization of the genetically altered proteins. The second aspect of this aim is to supplement the structure-function studies on HGPRT and XPRT via the introduction of crystallographic methods with the ultimate intention of resolving a 3-D structure of a leishmanial HGPRT or XPRT protein. The final specific aim is to ascertain the intracellular location of the APRT and XPRT proteins by subcellular fractionation of parasite lysates and by immunofluorescence and immunoelectron microscopy on intact cells.
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