Lu J Z, Muench S P, Allary M, Campbell S, Roberts C W, Mui E, McLeod R L, Rice D W, Prigge S T
Department of Molecular Microbiology & Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA.
Parasitology. 2007 Dec;134(Pt.14):1949-62. doi: 10.1017/S0031182007003319. Epub 2007 Aug 13.
Apicomplexan parasites of the genus Eimeria are the major causative agent of avian coccidiosis, leading to high economic losses in the poultry industry. Recent results show that Eimeria tenella harbours an apicoplast organelle, and that a key biosynthetic enzyme, enoyl reductase, is located in this organelle. In related parasites, enoyl reductase is one component of a type II fatty acid synthase (FAS) and has proven to be an attractive target for antimicrobial compounds. We cloned and expressed the mature form of E. tenella enoyl reductase (EtENR) for biochemical and structural studies. Recombinant EtENR exhibits NADH-dependent enoyl reductase activity and is inhibited by triclosan with an IC50 value of 60 nm. The crystal structure of EtENR reveals overall similarity with other ENR enzymes; however, the active site of EtENR is unoccupied, a state rarely observed in other ENR structures. Furthermore, the position of the central beta-sheet appears to block NADH binding and would require significant movement to allow NADH binding, a feature not previously seen in the ENR family. We analysed the E. tenella genomic database for orthologues of well-characterized bacterial and apicomplexan FAS enzymes and identified 6 additional genes, suggesting that E. tenella contains a type II FAS capable of synthesizing saturated, but not unsaturated, fatty acids. Interestingly, we also identified sequences that appear to encode multifunctional type I FAS enzymes, a feature also observed in Toxoplasma gondii, highlighting the similarity between these apicomplexan parasites.
艾美耳属的顶复门寄生虫是禽球虫病的主要病原体，给家禽业带来了巨大的经济损失。最近的研究结果表明，柔嫩艾美耳球虫含有一个顶质体细胞器，并且一种关键的生物合成酶——烯酰还原酶位于该细胞器中。在相关寄生虫中，烯酰还原酶是II型脂肪酸合酶（FAS）的一个组成部分，并且已被证明是抗菌化合物的一个有吸引力的靶点。我们克隆并表达了柔嫩艾美耳球虫烯酰还原酶（EtENR）的成熟形式，用于生化和结构研究。重组EtENR表现出依赖NADH的烯酰还原酶活性，并被三氯生抑制，IC50值为60纳米。EtENR的晶体结构显示出与其他ENR酶的总体相似性；然而，EtENR的活性位点是空的，这种状态在其他ENR结构中很少观察到。此外，中央β-折叠的位置似乎会阻碍NADH的结合，并且需要显著移动才能允许NADH结合，这是ENR家族中以前未见的特征。我们分析了柔嫩艾美耳球虫基因组数据库中特征明确的细菌和顶复门FAS酶的直系同源物，并鉴定出另外6个基因，这表明柔嫩艾美耳球虫含有一种能够合成饱和脂肪酸但不能合成不饱和脂肪酸的II型FAS。有趣的是，我们还鉴定出了似乎编码多功能I型FAS酶的序列，这一特征在刚地弓形虫中也有观察到，突出了这些顶复门寄生虫之间的相似性。
