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UMP synthesis in the kinetoplastida
Abstract:
All six enzymes of pyrimidine biosynthesis de novo have been detected in homogenates of the culture promastigote form of Leishmania mexicana amazonensis, the blood trypomastigote form of Trypanosoma brucei and the culture epimastigote, blood trypomastigote and intracellular amastigote forms of Trypanosoma cruzi. Dihydroorotate dehydrogenase is mitochondrial in mammals, but the isofunctional enzyme, dihydroorotate oxidase was found to be cytoplasmic, whereas orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase, which are cytoplasmic in mammals, were found to be particulate. Analysis by isopycnic sedimentation in sucrose showed that both particulate enzymes co-sedimented with glycosomal-(microbody-)marker enzymes such as hexokinase. Electron microscopy indicated that fractions containing these activities consisted essentially only of microbodies. It is concluded therefore that these enzymes are associated with glycosomes. Kinetic studies with intact glycosomal preparations suggested that there was no membrane barrier between 5-phosphoribose 1-pyrophosphate (P-Rib-PP) and orotate phosphoribosyltransferase, indicating either that the active site of this enzyme is probably on the outside of the glycosome or that the glycosome may have an efficient transport site for P-Rib-PP. Not all the UMP salvage enzymes assayed were detected. No uridine kinase activity was found in any of the species investigated, suggesting that uridine salvage might be routed via a uridine phosphorylase and uracil phosphoribosyltransferase. In agreement with this suggestion, these latter activities were detected in all organisms tested except the intracellular amastigote form of T. cruzi, where uracil phosphoribosyltransferase appeared absent. All the UMP salvage enzymes investigated occurred in cytoplamic fractions.
Insights
Pyrimidine biosynthesis enzymes are present in Leishmania and Trypanosoma parasites. Key enzymes are localized to glycosomes, suggesting potential drug targets for treating parasitic diseases.
Area of Science:
- Biochemistry
- Parasitology
- Molecular Biology
Background:
- Pyrimidine biosynthesis is essential for parasitic protozoa.
- Understanding enzyme localization is crucial for identifying drug targets.
Purpose of the Study:
- To investigate the localization of de novo pyrimidine biosynthesis and UMP salvage enzymes in Leishmania and Trypanosoma species.
- To determine the subcellular location of key enzymes involved in pyrimidine metabolism.
Main Methods:
- Enzyme activity assays in parasite homogenates.
- Subcellular fractionation using isopycnic sedimentation.
- Electron microscopy for organelle identification.
- Kinetic studies with isolated glycosomes.
Main Results:
- All six de novo pyrimidine biosynthesis enzymes were detected in Leishmania mexicana amazonensis and Trypanosoma species.
- Dihydroorotate oxidase, orotate phosphoribosyltransferase, and orotidine-5'-phosphate decarboxylase were found to be associated with glycosomes.
- Uridine kinase was absent, while uridine phosphorylase and uracil phosphoribosyltransferase activities were detected (except in T. cruzi amastigotes).
Conclusions:
- Key enzymes of de novo pyrimidine biosynthesis are localized to glycosomes in these parasites.
- The glycosomal localization of these enzymes suggests they are potential targets for antiparasitic drug development.
- The absence of uridine kinase points to alternative salvage pathways for pyrimidine synthesis.