Na+-pumping NADH:quinone oxidoreductase of V. cholerae

钠泵NADH:霍乱弧菌醌氧化还原酶

• 批准号: 6714192
• 负责人: Blanca Barquera
• 金额: $ 18.95万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-03 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6714192
• 关键词: NAD(P)H oxidoreductase; Vibrio cholerae; active sites; active transport; bioterrorism /chemical warfare; chemical kinetics; chimeric proteins; cofactor; computer simulation; crystallization; electrical property; electron transport; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; ion transport; membrane structure; oxidation reduction reaction; physical model; protein structure function; site directed mutagenesis; sodium ion; stop flow technique

DESCRIPTION (provided by applicant): The proposed research is focused on the structure and function of the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae. This enzyme is the primary gateway for electrons into the aerobic respiratory chain of many marine and pathogenic bacteria. As such it plays a role similar to Complex of the mitochondrial respiratory chain. However, Na+-NQR has no homology to Complex I, and instead of translocating protons, pumps sodium ions across the cell membrane creating a sodium motive force that is used by the cell for metabolic work. Sodium metabolism plays an important role in the adaptation of Vibrio cholerae to different environments encountered in its cycle of propagation and infection. Furthermore, Na+ -NQR has been implicated in the regulation of virulence factors in Vibrio cholerae. Our goal is to understand the mechanism by which redox reactions are harnessed to drive the translocation of sodium in Na+-NQR. For this, it is important to study both the redox processes and the mechanism of sodium transport. We will use an approach that combines site-directed mutagenesis with kinetics and other biophysical methods. We have developed a recombinant Na+-NQR in Vibrio cholerae, an organism that is congenial to genetic manipulation and for which the complete genome sequence is known. The recombinant enzyme is easily purified by means of a 6X-histidine-tag. We have already made several site-directed mutants that alter cofactor binding, demonstrating that this is a viable system to address functional questions. We plan to make additional mutants, including ones to target conserved charged and polar amino acid residues, which are likely to be involved in sodium pathways inside the enzyme. In order to design the mutants and to evaluate the results, we will need topological and structural information about the enzyme. To this end we plan to create membrane topology maps by using computer predictions together with reporter-protein fusion experiments. We will also make a strong effort to crystallize Na+-NQR, since a 3-dimensional structural model is essential for a molecular-level understanding of the mechanism of the enzyme.

描述（由申请人提供）：拟定研究的重点是来自霍乱弧菌的Na+泵NADH：醌氧化还原酶（Na+-NQR）的结构和功能。这种酶是电子进入许多海洋细菌和病原菌的有氧呼吸链的主要通道。因此，它起着类似于线粒体呼吸链复合体的作用。然而，Na+-NQR与复合物I没有同源性，并且不是使质子易位，而是将钠离子泵送穿过细胞膜，产生被细胞用于代谢工作的钠动力。钠代谢在霍乱弧菌适应其繁殖和感染周期中遇到的不同环境中起重要作用。此外，Na+ -NQR还参与了霍乱弧菌毒力因子的调节。 我们的目标是了解利用氧化还原反应来驱动Na+-NQR中钠的移位的机制。为此，研究氧化还原过程和钠转运机制是很重要的。我们将使用一种结合定点诱变与动力学和其他生物物理方法的方法。我们已经在霍乱弧菌中开发了重组Na+-NQR，霍乱弧菌是一种适合遗传操作的生物体，其完整的基因组序列是已知的。重组酶容易通过6X-组氨酸标签纯化。我们已经做了几个定点突变，改变辅因子结合，证明这是一个可行的系统，以解决功能问题。我们计划制造更多的突变体，包括针对保守的带电和极性氨基酸残基的突变体，这些氨基酸残基可能参与酶内的钠途径。为了设计突变体和评估结果，我们需要有关酶的拓扑和结构信息。为此，我们计划通过使用计算机预测和蛋白质融合实验来创建膜拓扑图。我们还将努力结晶Na+-NQR，因为三维结构模型对于分子水平上理解酶的机制至关重要。