INITIAL CONDITIONS FOR PARASITISM IN TRYPANOSOMES: APOSSIBLE ROLE OF ANAEROBIC PYRIMIDINE BIOSYNTHESIS

在本项目中，我们旨在验证锥虫从头合成嘧啶生物合成途径的进化意义，该途径包括六个顺序的酶促反应。该途径的第四个和第六个酶，二氢羟酸脱氢酶（DHOD）和欧罗嘌呤-5'-单磷酸脱羧酶（OMPDC）的基因先前已被证明具有侧基因转移（LGT）起源，突出了该途径在锥虫中的独特性质。研究了恰加斯病病原克氏锥虫DHOD的遗传多样性和动力学特性。克氏T. cruzi Tulahuen菌株的3个DHOD基因全长942 bp，差异26个核苷酸，导致8个氨基酸残基被替换。重组DHODs具有相似的酶学性质，包括底物和电子受体的最适pH、最适温度、V_<max>和K_m。这些结果表明，尽管存在遗传变异，但克氏T. cruzi DHODs的动力学特性是保守的。OMPDC基因与第五种酶——角状磷酸核糖基转移酶（OPRT）融合，在锥虫中定位于糖体中，而在动物和植物中则以OPRT-OMPDC融合的方式存在。基因克隆和系统发育分析表明，OMPDC-OPRT融合出现在由锥虫和齿类组成的动质体的共同祖先中，通过OPRT-OMPDC的分裂和再融合。OMPDC-OPRT融合也在另一个真核生物群——层堆中被发现。系统发育重建在统计上拒绝了层堆和着丝质体OMPDC-OPRT结构域的单一性，表明这些基因融合尽管具有相同的基因顺序，但并不具有共同的进化起源。因此，OMPDC-OPRT融合很可能在这些真核生物群体中独立出现。我们得出结论，基因融合事件发生的频率比以前认为的要高，LGT对真核生物中OPRT和OMPDC基因重排结构的建立做出了显著贡献。少

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 … More 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