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

钠泵NADH:霍乱弧菌醌氧化还原酶

• 批准号: 7942227
• 负责人: Blanca Barquera
• 金额: $ 7.59万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7942227
• 关键词: 3-Dimensional; ATP phosphohydrolase; Address; Aerobic; Alkaline Phosphatase; Amino Acids; Binding; Binding Sites; Biochemical; Biological Assay; Carboxy-Lyases; Cell membrane; Cells; Charge; Chemistry; Chimeric Proteins; Coenzymes; Complex; Computers; Coupled; Crystallization; Disputes; Electron Spin Resonance Spectroscopy; Electron Transport; Electrons; Environment; Enzymes; Evaluation; Flavins; Freezing; Generations; Genetic; Goals; Grant; Green Fluorescent Proteins; Histidine; Individual; Infection; Kinetics; Link; Liposomes; Maps; Marines; Membrane; Membrane Potentials; Metabolic; Metabolism; Methods; Methyltransferase; Mitochondria; Modeling; Molecular; Monitor; Motor; NADH; NADH dehydrogenase (quinone); Na(+)-K(+)-Exchanging ATPase; Organism; Oxalates; Oxidation-Reduction; Pathway interactions; Play; Process; Property; Proteins; Proton Pump; Pump; Quinones; Reaction; Recombinants; Regulation; Reporter; Research; Respiratory Chain; Role; Site; Site-Directed Mutagenesis; Sodium; Structural Models; Structure; Structure-Activity Relationship; System; Testing; Titrations; Transport Reaction; Vibrio cholerae; Virulence Factors; Work; citrate carrier; cofactor; design; driving force; enzyme mechanism; genetic manipulation; genome sequencing; interest; millisecond; mutant; next generation; pathogenic bacteria; reconstitution; research study; respiratory; sodium ion; symporter

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）：拟议的研究集中在Na+泵送NADH的结构和功能上：来自Vibrio Cholerae的喹酮氧化还原酶（Na+-NQR）。该酶是将电子进入许多海洋和致病细菌的有氧呼吸链的主要门户。因此，它起着类似于线粒体呼吸链复合物的作用。但是，Na+-NQR与复合物I没有同源性，而不是易位的质子，而是在细胞膜上泵送钠离子，产生了一个由细胞用于代谢工作的钠动力。钠代谢在适应弧菌霍乱的传播和感染周期中遇到的不同环境中起着重要作用。此外，Na+ -NQR已与Vibrio Cholerae中的毒力因子的调节有关。 我们的目标是了解利用氧化还原反应驱动Na+-NQR中钠易位的机制。为此，重要的是研究氧化还原过程和钠转运的机制。我们将使用一种将定位诱变与动力学和其他生物物理方法相结合的方法。我们已经在Vibrio Cholerae中开发了一个重组Na+-NQR，这是一种对遗传操作的友善的生物体，并且已知完整的基因组序列。重组酶很容易通过6倍齐丁烷标记纯化。我们已经制造了几个位于站点定向的突变体，以改变辅因子的结合，这表明这是解决功能问题的可行系统。我们计划制造其他突变体，包括靶向保守的电荷和极性氨基酸残基，这些突变体可能与酶内部的钠途径有关。为了设计突变体并评估结果，我们将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以及记者 - 蛋白融合实验来创建膜拓扑图。我们还将做出巨大的努力来结晶Na+-NQR，因为三维结构模型对于对酶机制的分子级别的理解至关重要。

项目成果

Covalent binding of flavins to RnfG and RnfD in the Rnf complex from Vibrio cholerae.

• DOI: 10.1021/bi800920j
• 发表时间: 2008-10-28
• 期刊: BIOCHEMISTRY
• 影响因子: 2.9
• 作者: Backiel, Julianne;Juarez, Oscar;Zagorevski, Dmitri V.;Wang, Zhenyu;Nilges, Mark J.;Barquera, Blanca
• 通讯作者: Barquera, Blanca