The goal of the proposed research is to gain a better understanding of pyrimidine biosynthesis in Toxoplasma gondii. The presence of the pathway in this organism has been established (Schwartzman & Pefferferkorn, 1981; Asai et al., 1983), however none of the enzymes involved have been fully characterized or cloned. We will study dihydroorotase (DHOase) and dihydroorotate dehydrogenase (DHODase) which catalyze the third and fourth reactions, respectively. Large quantities of recombinant enzymes will be produced in bacterial or yeast expression systems. We will determine whether T.gondii DHOase is a monofunctional enzyme or a domain of a multifunctional enzyme. Our studies will show whether the membrane- associated T. gondii DHODase is a simple flavoprotein, as are some protozoan DHODases (Pascal et al., 1983), or whether it is a two-cofactor protein like the bovine DHODase (Hines & Johnston, 1989). Analysis of sequences, kinetic characteristics, and metal/cofactor composition of T. gondii DHOase and DHODase will lead to an understanding of the catalytic mechanisms at a molecular level. A comparison of the enzymes with their mammalian counterparts will reveal whether T. gondii DHOase and DHODase might be appropriate targets for compounds which could serve as drugs. Crystallization and structural analysis of T. gondii DHOase will allow identification of active site residues and will be useful for identifying such residues in other DHOases. Structural information will be invaluable in understanding the efficacy of inhibitors, and will aid in identifying other inhibitory compounds. We will determine whether DHODase binds to coenzyme Q homologs and hydroxynaphthoquinones which are active against T. gondii (Ellis, 1994; Araujo et al, 1991). Future directions of the proposed research are to investigate the role of individual residues in the recombinant enzymes by site-directed mutagenesis, to conduct studies with DHOase and DHODase inhibitors which have anti-cancer or antimalarial activity, to investigate potential co-regulation of purine salvage and pyrimidine synthesis, and to investigate linkage of pyrimidine biosynthesis and the election transport chain.
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