PURINE/PYRIMIDINE METABOLISM IN TRICHOMONAS AND GIARDIA

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Trichomoniasis and giardiasis are two prevalent parasitic diseases in the world. The lack of effective chemoprophylactic and chemotherapeutic controls of them today are of great concern. We have recently indicated that Trichomonas and Giardia are both incapable of de novo synthesis of purine and pyrimidine nucleotides, which may constitute a major vulnerability for them. Several major, indispensable salvage enzymes have since been identified in these parasites, among which T. foetus IMP dehydrogenase, T. foetus hypoxanthanine-guanine-xanthine phosphoribosyltransferase and G. lamblia guanine phosphoribosyltransferase have been each purified to homogeneity and characterized. All three enzymes demonstrated unique kinetic properties and distinctive substrate specificities which may quality them as the chemotherapeutic targets. Further studies of the kinetic isotope effects and the isotope exchange on T. foetus IMP dehydrogenase will be performed for more understanding on the mechanism of the enzyme-catalyzed reaction and the unusual properties of its dinucleotide binding site. Genes encoding these three enzymes will be identified by polymerase chain reactions, cloned, sequenced and expressed in bacteria, yeast, mammalian cells or baculovirus for high yields of native proteins. The recombinant enzymes thus produced and purified will be further analyzed for detailed mechanisms of enzyme catalysis. Their primary structures derived from the encoding DNA sequences will be analyzed by computer modelings coupled with site-directed mutagenesis for elucidation of tertiary protein structures. In the long-term future plan, pure proteins will be crystallized for X-ray diffraction analysis of the three dimensional structures and comparisons among the protein structures for structure-function relationships for specific inhibitor designs. A hypoxanthine/guanine transporter was recently identified in T. foetus. It is apparently essential for the survival of this parasite in its wild-type. We will try to identify the transporter by inhibitor studies and isolate this transporter by photoaffinity labeling and develop it as a new target of anti-trichomonial chemotherapy for the future.