Parasitic protozoa are a heterogeneous group of unicellular organisms causing diseases such as malaria, Chagas diseases, and toxoplasmosis, which affect millions of people world wide. The agent causing toxoplasmosis, Toxoplasma gondii, produces life-threatening infections in immunosuppressed patients and in the fetus (Wong and Remington, 1993; Wong and Remington, 1994). Toxoplasmic encephalitis occurs in 10-32% of HIV-positive patients in the United States (Ammassari et al, 1996). The proposed research focuses on two enzymes of the de novo pyrimidine biosynthetic pathway in T. gondii. The aspartate transcarbamylase (ATCase) will be studied to generate basic information about the structure of the pathway. Sequencing the ATCase will show whether multi- functional or mono-functional proteins catalyze the first three steps. Kinetic analysis will determine whether the step catalyzed by ATCase is regulated. The proposed research would produce the first recombinant ATCase from a protozoan parasite, and would allow crystallization trials to take place. The ubiquinone binding site of the dihydroorotate dehydrogenase (DHODase) will be used as a model to test whether the species specificity for ubiquinones of a given polyprenyl chain length is a feature of such sites. Residues in the sites will be identified using photoreactive ubiquinone analogs. Site-directed mutagenesis will be conferred by making mutations at the site. These studies may shed light on why ubiquinone analogs, such as atovaquone, exhibit species specificity. Atovaquone is currently used as an alternative treatment for HIV-associated toxoplasmosis (Fung and Kirschenbaum, 1996), and is in clinical trials in patients with Plasmodium flaciparum malaria (Lell et al., 1998).
Showing the most recent 10 out of 174 publications
