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

锥虫中参与嘧啶从头生物合成的所有六种酶及其相应基因的分子特征

基本信息

批准号：
12470061
负责人：
AOKI Takashi
金额：
$ 8.51万
依托单位：
Juntendo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

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

最近，我们报道了存在的嘧啶生物合成（pyr）基因簇作为一个多顺反子转录单位，其中包含pyr 1，pyr 3，pyr 6/5，pyr 2，和pyr 4编码的所有六种酶的途径，在克氏锥虫。墨西哥利什曼原虫也有一个同源的pyr基因簇。这六种利什曼原虫酶类似于相应的T. cruzi同行;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）可能通过水平基因转移获得，共价连接的第6/5个酶（OPRT）具有C末端SKL基序，SKL基序是进入糖体（锥虫特有的细胞器）的必需靶向信号。大肠杆菌中，亲和纯化，酶活性以乳清酸生产量的形式进行测定。褐藻、墨角藻和Pelvetia的粗甲醇提取物 ...更多信息在50 μ g/ml的浓度下，巴宾顿菌的DHOD活性分别降低了50%和70%。动力学分析表明，这些提取物对寄生虫酶的作用模式是非竞争性的相对于底物，二氢乳清酸。为了评价这两种提取物对感染率的影响，测定了T。cruzi，将EtOH重构的样品加入体外感染系统，T. cruzi-HeLa细胞培养物。浓度为10 μ g/ml的两种样品均将感染率显著降低至对照的约40%。这些结果表明F. evanescens和P. babingtonii含有抑制T. cruzi DHOD活性和宿主细胞感染率的关系。进一步筛选其它海藻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。Cruzi在感染的HeLa细胞中生长更快，培养基中出现更多数量的锥鞭毛体。结果表明，CPS II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。对T. cruzi OPRT和OMPDC非常有趣，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）