Molecular characterization of all the six enzymes and their corresponding genes involved in de novo pyrimidine biosynthesis in trypanosomatids

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Recently, we reported the presence of the pyrimidine-biosynthetic (pyr) gene cluster as a polycistronic transcription unit, that contained pyrl, pyr3, pyr6/5, pyr2, and pyr4 encoding all the six enzymes of the pathway, in Trypanosoma cruzi. Leishmania mexicana also had a homologous pyr gene cluster. The six Leishmania enzymes resembled the corresponding T. cruzi counterparts; the first 3 enzymes (CPS II, ACT, DHO) may have resulted from early eukaryotic ancestors, the fourth (DHOD) and sixth (OMPDC) enzymes may have been acquired by horizontal gene transfers, and the covalently linked sixth/fifth (OPRT) enzymes possess a C-terminal SKL motif, an essential targeting signal into the glycosome (a peculiar organelle in trypanosomatids).The previously established recombinant DHOD was expressed in E. coli, affinity-purified, and the enzyme activity was measured as the amount of orotate production spectrophotometrically. Crude methanol-extracts from brown algae, Fucus evanescent and Pelvetia … More babingtonii, showed 50 and 70% reduction in the DHOD activity, respectively, at the concentration of 50 micro g/ml. Kinetic analysis exhibited that the mode of action of these extracts on the parasite enzyme was non-competitive with respect to the substrate, dihydroorotate. To estimate the effects of these two extracts on the infection rate, the percentage of host cells infected by T. cruzi, EtOH- reconstituted samples were added to the in vitro infection system, T. cruzi-HeLa cell cultures. Both samples, at the concentration of 10 micro g/ml, markedly lowered the rate of infection to approximately 40% of the control. These results imply that F. evanescens and P. babingtonii contain substances inhibitory to the T. cruzi DHOD activity and to the infection rate of host cells. Further screening of other marine algae and purification of the effective compound(s) may facilitate the discovery of a new, anti-trypanosomal lead compoundFurther, we examined effect of the trypanosomal CPS II overproduction in the protozoan using a shattle vector pTEX harboring pyrl; the transformed T. cruzi grew faster in the infected HeLa cells and a greater number of trypomastigotes appeared in the culture medium. The results suggest that a higher level of expression of CPS II. The rate-limiting enzyme of the pyrimidine-biosynthetic pathway, may have resulted in a higher level of pathogenicity, namely, faster growth. Further characterization of the T. cruzi OPRT and OMPDC is of great interest, particularly with respect to the comparison with the Plasmodium enzymes (because of the P. falciparum genome project completed) Less