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

最近，我们报道了在克氏锥虫中存在嘧啶生物合成（pyr）基因簇作为多顺反子转录单元，包含pyrl， pyr3, pyr6/5， pyr2和pyr4，编码该途径的所有六种酶。墨西哥利什曼原虫也有一个同源的pyr基因簇。6种利什曼原虫酶与相应的克氏T.相似；前3个酶（CPS II、ACT、DHO）可能来自早期真核生物祖先，第4个酶（DHOD）和第6个酶（OMPDC）可能是通过水平基因转移获得的，共价连接的第6 / 5个酶（OPRT）具有c端SKL基序，这是进入糖体（锥虫特有的细胞器）的必要靶向信号。先前建立的重组DHOD在大肠杆菌中表达，进行亲和纯化，并用分光光度法测定酶活性。褐藻粗甲醇提取物和褐藻褐角藻粗甲醇提取物在浓度为50微g/ml时，DHOD活性分别降低50%和70%。动力学分析表明，这些提取物对寄生虫酶的作用方式与底物二氢旋酸酯不具有竞争性。为了评估这两种提取物对感染率的影响，将重组的克氏t细胞感染的百分比加入体外感染系统克氏t - hela细胞培养中。在浓度为10微g/ml时，两种样品的感染率均显著降低，约为对照组的40%。这些结果表明，叶氏假单胞菌和巴丁氏假单胞菌含有抑制克氏T. DHOD活性和抑制宿主细胞感染率的物质。进一步筛选其他海洋藻类和纯化有效化合物可能有助于发现新的抗锥虫先导化合物。此外，我们使用携带pyrl的梭形载体pTEX检测了锥虫CPS II在原生动物体内过量产生的影响；转化后的克氏锥虫在感染的HeLa细胞中生长更快，培养基中出现了更多的锥乳线虫。结果表明，CPSⅱ的表达水平较高。嘧啶-生物合成途径的限速酶可能导致更高水平的致病性，即更快的生长。克氏疟原虫OPRT和OMPDC的进一步表征是非常有趣的，特别是与疟原虫酶的比较（因为恶性疟原虫基因组计划已经完成）

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