Plasmodium falciparum acyl carrier protein crystal structures in disulfide-linked and reduced states and their prevalence during blood stage growth.
Plasmodium falciparum acyl carrier protein crystal structures in disulfide-linked and reduced states and their prevalence during blood stage growth.
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DOI：
10.1002/prot.22582
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发表时间：
2010-02-15
影响因子：
2.9
通讯作者：
Prigge, Sean T.
中科院分区：
文献类型：
--
作者：
Gallagher, John R.;Prigge, Sean T.
关键词：
酰基载体蛋白（ACP）具有在生物合成期间共价结合新生脂肪酸的功能所必需的单个反应性巯基。在恶性疟原虫中，最致命形式的疟疾的病原体，在寄生虫生命周期的肝脏阶段期间，脂肪酸生物合成发生在顶质体细胞器中。在血液阶段，脂肪酸的生物合成是不活跃的，顶质体的氧化还原状态尚未确定。我们解析了还原和二硫键连接形式的来自恶性疟原虫的ACP的晶体结构，并观察到令人惊讶的结果，即PfACP交联二聚体中的二硫键在紧密的分子界面中与本体溶剂隔离。我们用小分子还原剂评估了二硫化物的溶剂可及性，发现二硫化物受到BME的保护，但其他常见还原剂的保护较少。我们检查了培养的恶性疟原虫寄生虫，以确定哪种形式的PfACP在血液阶段是普遍的。我们很容易检测到单体PfACP寄生虫裂解液中，但没有观察到二硫键连接的形式，即使在氧化应激的条件下。为了证明PfACP含有游离巯基并且未酰化或处于脱辅基状态，我们用二硫化物形成试剂二酰胺处理血液期寄生虫。我们发现，二酰胺的影响与还原剂逆转。总之，这些结果表明，顶质体是一个还原室，如恶性疟原虫代谢模型所示，PfACP在血液阶段生长期间维持在还原状态。
Acyl Carrier Protein (ACP) has a single reactive sulfhydryl necessary for function in covalently binding nascent fatty acids during biosynthesis. In Plasmodium falciparum, the causative agent of the most lethal form of malaria, fatty acid biosynthesis occurs in the apicoplast organelle during the liver stage of the parasite life cycle. During the blood stage, fatty acid biosynthesis is inactive and the redox state of the apicoplast has not been determined. We solved the crystal structure of ACP from P. falciparum in reduced and disulfide-linked forms, and observe the surprising result that the disulfide in the PfACP cross-linked dimer is sequestered from bulk solvent in a tight molecular interface. We assessed solvent accessibility of the disulfide with small molecule reducing agents and found that the disulfide is protected from BME but less so for other common reducing agents. We examined cultured P. falciparum parasites to determine which form of PfACP is prevalent during the blood stages. We readily detected monomeric PfACP in parasite lysate, but do not observe the disulfide-linked form, even under conditions of oxidative stress. To demonstrate that PfACP contains a free sulfhydryl and is not acylated or in the apo state, we treated blood stage parasites with the disulfide forming reagent diamide. We found that the effects of diamide are reversed with reducing agent. Together, these results suggest that the apicoplast is a reducing compartment, as suggested by models of P. falciparum metabolism, and that PfACP is maintained in a reduced state during blood stage growth.
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影响因子：
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作者：
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DOI：
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