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

钠泵NADH:霍乱弧菌醌氧化还原酶

• 批准号: 7479334
• 负责人: Blanca Barquera
• 金额: $ 16.01万
• 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 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7479334
• 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; Helix (Snails); Histidine; Individual; Infection; Kinetics; Link; Liposomes; Localized; Maps; Marines; Membrane; Membrane Potentials; Metabolic; Metabolism; Methods; Methyltransferase; Mitochondria; Modeling; Molecular; Monitor; Motor; Mutagenesis; NADH; NADH dehydrogenase (quinone); 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; benzoquinone; citrate carrier; cofactor; design; driving force; ear helix; 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）：所提议的研究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细菌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体的作用。然而，Na+-NQR 与复合物 I 没有同源性，并且不是易位质子，而是泵送钠离子穿过细胞膜，产生钠动力，细胞利用该动力进行代谢工作。钠代谢在霍乱弧菌适应其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 与霍乱弧菌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，研究氧化还原过程和钠转运机制非常重要。我们将使用一种将定点诱变与动力学和其他生物物理方法相结合的方法。我们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，并且其完整基因组序列已知。重组酶可通过 6X-组氨酸标签轻松纯化。我们已经制造了几种改变辅因子结合的定点突变体，证明这是解决功能问题的可行系统。我们计划制造更多突变体，包括针对保守带电和极性氨基酸残基的突变体，这些残基可能参与酶内的钠途径。为了设计突变体并评估结果，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报告蛋白融合实验来创建膜拓扑图。我们还将努力结晶 Na+-NQR，因为 3 维结构模型对于分子水平理解酶的机制至关重要。