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

描述(申请人提供)：建议的研究重点是霍乱弧菌钠泵NADH：苯醌氧化还原酶(Na-NQR)的结构和功能。这种酶是电子进入许多海洋和致病细菌需氧呼吸链的主要通道。因此，它的作用类似于线粒体呼吸链的复合体。然而，Na-NQR与络合物I没有同源性，它不是转移质子，而是将钠离子泵过细胞膜，产生钠动力，被细胞用于新陈代谢工作。钠代谢在霍乱弧菌在其繁殖和感染周期中对不同环境的适应中起着重要的作用。此外，Na-NQR还参与霍乱弧菌毒力因子的调节。 我们的目标是了解氧化还原反应被利用来驱动钠在Na-NQR中转移的机制。为此，研究氧化还原过程和钠的转运机制是非常重要的。我们将使用一种将定点突变与动力学和其他生物物理方法相结合的方法。我们已经在霍乱弧菌中开发了一种重组的Na-NQR，霍乱弧菌是一种适合遗传操作的生物，其完整基因组序列已知。重组酶可通过6X-组氨酸标签进行纯化。我们已经制作了几个改变辅因子结合的定点突变体，证明这是一个解决功能问题的可行系统。我们计划制造更多的突变体，包括针对保守的带电氨基酸和极性氨基酸残基的突变，这些氨基酸可能参与酶内的钠通路。为了设计突变体并评估结果，我们需要关于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。我们还将努力使Na-NQR结晶，因为三维结构模型对于在分子水平上理解酶的机制是必不可少的。

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.