Studies on Molecular Biology of Prostaglandin Biosynthesis by parasitic Trypanosoma

本研究的目的是阐明寄生原虫（锥虫属、利什曼原虫属和疟原虫属）前列腺素（PG）合成酶的功能和三维结构，并开发抗寄生虫药物。布氏锥虫前列腺素（PG）F_（2α）合酶（TbPGFS）是一种醛酮还原酶（AKR），催化PGH_2的内过氧化物部分还原为PGF_（2α），PGF_（2α）在锥虫感染时过量产生，导致流产。本文报道了TbPGFS与NADP^+和柠檬酸盐复合物的晶体结构，分辨率为2.1 nm。TbPGFS为平行的（αβ）_8桶形褶皱，缺乏双曲环。核心活性位点结构是疏水性的以结合疏水底物，并且含有酪氨酸、赖氨酸、组氨酸和天冬氨酸，在其他醛酮还原酶中被称为催化四联体。突变鉴定表明Tyr 52和Asp 47与酶反应无关，表明His 110和Lys 77形成催化二联体进行酸辅助还原，其中His 110将其质子转移到底物。这些发现为醛酮还原酶生物还原内过氧化物PGH_2提供了一种新的催化机制。该结构应允许合理设计的特定抑制剂，用于调查TbPGFs在锥虫中的生理作用。PGH_2的还原作用已为人所知，也有研究表明PGF_（2α）是通过PGH_2 α的9，11-内过氧化物的还原裂解而酶促合成的<43>。然而，该酶如何进行PGH_2的9，11-内过氧化物的还原性裂解尚未阐明。本文首次报道了原生动物PGH_2还原酶的结构信息。这种结构现在应该允许合理设计的具体抑制剂，可能有助于调查TbPGFs在锥虫的生理作用。

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.