| Research Abstract |
In this project, we aimed to verify evolutionary implications of de novo pyrimidine biosynthetic pathway, which includes six sequential enzymatic reactions, in trypanosomes. Genes for the fourth and sixth enzymes of the pathway, dihydroorotate dehydrogenase (DHOD) and orotidine-5'-monophosphate decarboxylase (OMPDC), respectively, have been previously shown to have a lateral-gene-transfer (LGT) origin, highlighting the distinct nature of the pathway in trypanosomes. We demonstrated that genetic diversity and the kinetic properties of DHOD in Trypanosoma cruzi, the agent of Chagas' disease. Three DHOD genes in T. cruzi Tulahuen strain were comprised of 942 bp and differed by 26 nucleotides, resulting in replacement of 8 amino acid residues. Recombinant DHODs showed similar enzymatic properties, including optimal pH, optimal temperature, V_<max>, and K_m for the substrate and electron acceptor. These results suggest that, despite their genetic variations, kinetic properties of the three
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T. cruzi DHODs are conserved.
OMPDC gene is fused with the fifth enzyme, orotate phosphoribosyltransferase (OPRT) as OMPDC-OPRT fusion and localized in glycosomes in trypanosomes, while they occur as OPRT-OMPDC fusion in animals and plants. Gene cloning and phylogenetic analyses suggested that OMPDC-OPRT fusion emerged in a common ancestor of kinetoplastids that comprises trypanosomatids and bodonids, via split of OPRT-OMPDC and re-fusion. OMPDC-OPRT fusion was also found in another eukaryotic group, stramenopiles. Phylogenetic reconstruction rejected statistically the monophyly of the OPRT domains of stramenopile and kinetoplastid OMPDC-OPRT, demonstrating that these gene fusions do not share a common evolutionary origin, despite the identical gene order. Thus, the OMPDC-OPRT fusion is likely to have emerged independently in these eukaryotic groups. We conclude that gene fusion events occur more frequently than previously thought and that LGT has made a marked contribution to establishment of the rearranged structure of OPRT and OMPDC genes in eukaryotes. Less
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