Development ofenzyme inlulkar far infectious diseace including malaria and clarification ofthe inlubitor activity

人类疟疾、利什曼病、恰加斯病、非洲人类锥虫病、登革热和血吸虫病等热带疾病继续造成大量发病率和死亡率。在本项目中，我们对这三种传染病的寄生虫中重要的酶进行了x射线晶体学研究，以构建用硅内法开发药物的结构基础。重要的酶是：(1)两种与人疟疾中嘧啶核苷酸从头合成有关的酶，(2)来自恰加斯病和利什曼原虫的前列腺素（PG） F合成酶。从大肠杆菌中分离纯化了重组酶——欧罗替丁磷酸核糖基转移酶（pyr5）和欧罗替丁5′-单磷酸脱羧酶（pyr6）。结果，对pyr6的原生和产物UMP配合物进行了结晶，并进行了x射线结构分析。载脂蛋白和复合物形式pyr6的2.7和2.6 A分辨率结构提供了脱羧机制的细节(J. Biochem。, 2008)。对克氏锥虫和大利什曼原虫的PGF合成酶也进行了结晶，并在1.7 A分辨率下测定了克氏锥虫PGF合成酶的结构（Acta crystal）。F, 2007)。测定了PGF合酶与吲哚美辛的复合物结构，为开发新的抑制剂提供了结构信息。

Tropical diseases such as human malaria, leishmaniasis, Chagas disease, human African trypanosomiasis, dengue fever and schistosomiasis continue to cause significant morbidity and mortality. In this project, we performed X-ray crystallographic studies of important enzymes from parasite concerning three these infectious disease to construct the structural basis for the drug development by the in-silica method. The important enzymes are (1) two kinds of enzymes concerning the de novo synthesis of pyrimidine nucleotide from human malaria, (2) prostaglandin (PG) F synthases from parasites of Chagas' disease and Leishmania.Recombinant enzymes of orotate phosphoribosyltransferase (pyr5) and Orotidine 5'-monophosphate decarboxylase (pyr6) were purified from E.coli and crystallized. As the result, the native and product UMP complex form of pyr6 were crystallized and carried out with the X-ray structural analyses. The 2.7 and 2.6 A resolutional structures of apo- and complex forms of pyr6 provided the details of the decarboxylation mechanism (J. Biochem., 2008). As for the PGF synthases from Trypanosoma cruzi and Leishmania mejor were also performed crystallization and the structure of PGF synthase from T. cruzi were determined at 1.7 A resolution (Acta Cryst. F, 2007). The complex structure of PGF sytnthase with indomethacin were also determined, providing the structural information for new inhibitor development.