Studies on Molecular Biology of Prostaglandin Biosynthesis by parasitic Trypanosoma

本研究旨在阐明前列腺素(PG)合成酶在寄生性原虫(锥虫、利什曼原虫和疟原虫)中的功能和三维结构，以及抗寄生虫药物的开发。布氏锥虫前列腺素(PG)F&gt；2α&gt；合成酶(TbPGFS)是一种醛酮还原酶，它催化依赖NADPH的PGH_2内过氧化部分还原为PGF_2α&gt；本文报道了TbPGFS与NaDP和柠檬酸形成的络合物的晶体结构，分辨率为2.1Å。TbPGFS采用平行的(αβ)_8桶褶皱，没有凸起的环。核心活性中心结构是疏水的，结合疏水底物，含有酪氨酸、赖氨酸、组氨酸和天冬氨酸，在其他醛酮还原酶中称为催化四联体。突变鉴定发现Tyr 52和Asp 47与酶反应无关，表明His 110和Lys 77形成催化二聚体，执行酸辅助还原，His 110将其质子转移到底物。这些发现确定了醛酮还原酶生物还原内过氧物PGH_2的一种新的催化机理。这种结构应该允许合理设计特定的抑制剂，有助于研究TbPGFS在锥体中的生理作用。前列腺素H_2的还原已知一段时间，也有研究表明，前列腺素F_2；2α&gt；是由PGH_2^&t；43&gt；的9，11-内源性过氧化物酶催化裂解合成的。然而，该酶如何作用于PGH_2的9，11-内源性过氧化产物的还原裂解还没有被阐明。在这里，我们提供了原生动物酶中PGH_2还原酶的第一个结构信息。这种结构现在应该允许合理设计特定的抑制剂，这可能有助于研究TbPGFS在锥体中的生理作用。

In this study our aims are the elucidation of the functions and the three-dimensional structures of prostaglandin (PG) synthetic enzymes in parasitic protozoa (Trypanosoma, Leishmania, and Plasmodia), and the development of anti-parasitic drugs. Trypanosoma brucei prostaglandin (PG) F_<2α> synthase (TbPGFS), an aldo-ketoreductase (AKR), catalyzes the NADPH-dependent reduction of the endoperoxide moiety of PGH_2 into PGF_<2α> whose overproduction during trypanosomiasis causes abortion in infected subjects. Here we report the crystal structure of TbPGFS in complex with NADP^+ and citrate at 2.1 Å resolution. TbPGFS adopts a parallel (αβ)_8-barrel fold lacking the protrudent loops. The core active site structure is hydrophobic to bind hydrophobic substrates and contains tyrosine, lysine, histidine and aspartate known as a catalytic tetrad in other aldo-ketoreductases. Mutagenesis identifies that Tyr 52 and Asp 47 are not concerning to the enzyme reaction, showing His 110 and Lys 77 forms the catalytic dyad to perform an acid-assisted reduction in which His 110 transfers its proton to the substrate. These findings identify a novel catalytic mechanism for the biological reduction of the endoperoxide PGH_2 by an aldo-ketoreductase. The structure should allow for rational design of specific inhibitors useful to investigate the physiological roles of TbPGFS in trypanosomes. The reduction of PGH2 has been known for some time and it has also been shown that PGF_<2α> is enzymatically synthesized by a reductive cleavage of the 9, 11-endoperoxide of PGH_2^<43>. However, how the enzyme operates the reductive cleavage of the 9, 11-endoperoxide of PGH_2 has not been elucidated. We provide here the first structural information of a PGH_2 reductase in protozoa enzymes. This structure should now allow the rational design of specific inhibitors that might help investigate the physiological role of TbPGFS in trypanosomes.