Molecular organization and physiological functions of the respiratory chain specific for mitochondria from Plasmodium

疟原虫线粒体呼吸链的分子组织和生理功能

基本信息

批准号：
14021014
负责人：
KITA Kiyoshi
金额：
$ 36.48万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2005
项目状态：
已结题

项目摘要

1. Studies on the respiratory chain specific for mitochondria from PlasmodiumThe mitochondrion and the apicoplast of the malaria parasite, Plasmodium spp is microscopically observed in a close proximity to each other. In this study, we tested the suitability of two different separation techniques-Percoll density gradient centrifugation and fluorescence-activated organelle sorting for improving the purity of mitochondria isolated from the crude organelle preparation of P falciparum. To our surprise, the apicoplast was inseparable from the plasmodial mitochondrion by each method. This implies these two plasmodial organelles are bound each other. This is the first experimental evidence of a physical binding between the two organelles in Plasmodium.2. Studies on cyanide-resistant alternative oxidases from parasitic protozoa and its spescific inhibitor, ascofuranoneBased on amino acid sequence similarity and the ability to catalyze the four-electron reduction of oxygen to water using a quin … More ol substrate, mitochondrial alternative oxidase (AOX) and plastid terminal oxidase (PTOX) appear to be two closely related members of the membrane-bound diiron carboxylate group of proteins. In the current studies, we took advantage of the high activity of Trypanosoma vivax AOX (TvAOX) to examine the importance of the conserved Glu and the Tyr residues around the predicted third helix region of AOXs and PTOXs. We first compared the amino acid sequences of TvAOX with AOXs and PTOXs from various taxa and then performed alanine-scanning mutagenesis of TvAOX between amino acids Y199 and Y247. We found that the ubiquinol oxidase activity of TvAOX is completely lost in the E214A mutant, whereas mutants E215A and E216A retained more than 30% of the wild-type activity. Among the Tyr mutants, a complete loss of activity was also observed for the Y221A mutant, whereas the activities were equivalent to wild-type for the Y199A, Y212A, and Y247A mutants. Finally, residues G1u214 and Tyr221 were found to be strictly conserved among AOXs and PTOXs. Based on these findings, it appears that AOXs and PTOXs are a novel subclass of diiron carboxylate proteins that require the conserved motif E(X)eY for enzyme activity. Less

1. 疟原虫线粒体呼吸链的研究在显微镜下观察到疟原虫的线粒体和顶质体彼此非常接近，在这项研究中，我们测试了两种不同分离技术 - Percoll 密度的适用性。密度梯度离心和荧光激活细胞器分选可提高从恶性疟原虫细胞器粗制品中分离出的线粒体的纯度。通过每种方法，顶端质体与疟原虫线粒体密不可分，这意味着这两种疟原虫细胞器彼此结合，这是疟原虫中两种细胞器之间物理结合的第一个实验证据。2．原生动物及其特异性抑制剂壳二呋喃酮基于氨基酸序列相似性和催化能力使用 quin 底物将氧四电子还原为水，线粒体替代氧化酶 (AOX) 和质体末端氧化酶 (PTOX) 似乎是目前膜结合二铁羧酸蛋白组的两个密切相关的成员。研究中，我们利用间日锥虫 AOX (TvAOX) 的高活性来检查预测的第三螺旋区域周围保守的 Glu 和 Tyr 残基的重要性。我们首先将 TvAOX 与来自不同分类群的 AOX 和 PTOX 的氨基酸序列进行比较，然后对氨基酸 Y199 和 Y247 之间的 TvAOX 进行丙氨酸扫描诱变，我们发现 TvAOX 的泛醇氧化酶活性完全丧失。 E214A突变体，而突变体E215A和E216A保留了超过30%的在 Tyr 突变体中，还观察到 Y221A 突变体的活性完全丧失，而 Y199A、Y212A 和 Y247A 突变体的活性与野生型相同。最后，发现了残基 G1u214 和 Tyr221。 AOX 和 PTOX 之间严格保守基于这些发现，AOX 和 PTOX 似乎是二铁的一个新亚类。需要保守基序 E(X)eY 来实现酶活性的羧酸蛋白。

项目成果

期刊论文数量（68）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Mutational analysis of the Trypanosoma vivax alternative oxidase : the E(X)eY motif is conserved in both mitochondrial alternative oxidase and plastid terminal oxidase and is indispensable for enzyme activity

间日锥虫替代氧化酶的突变分析：E(X)eY基序在线粒体替代氧化酶和质体末端氧化酶中都是保守的，并且对于酶活性是不可或缺的

DOI：
发表时间：
2005
期刊：
Biochem. Biophys. Res. Commun. 334
影响因子：
0
作者：
Nakamura;K.;Sakamoto;K.Kido;Y.;Fujimoto;Y.;Suzuki;T.;Suzuki;M.;Yabu;Y;Ohta;N.;Tsuda;A.;Onuma;M.;Kita;K.
通讯作者：
K.

Up-regulation of Heme Biosynthesis during Neuronal Differentiation.

神经元分化过程中血红素生物合成的上调。