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

描述（由申请人提供）：拟研究的重点是来自霍乱弧菌的Na+泵送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致病菌有氧呼吸链的主要通道。因此，它的作用类似于线粒体呼吸链复合体。然而，Na+-NQR与复合体I没有同源性，它不是转运质子，而是将钠离子泵过细胞膜，产生钠动力，用于细胞的代谢工作。钠代谢在霍乱弧菌的繁殖和感染周期中对不同环境的适应中起着重要作用。此外，Na+ -NQR还参与了霍乱弧菌毒力因子的调控。

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.