Purification and characterization of the recombinant Na+-translocating NADH:quinone oxidoreductase from vibrio cholerae
Purification and characterization of the recombinant Na+-translocating NADH:quinone oxidoreductase from vibrio cholerae
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DOI：
10.1021/bi011873o
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发表时间：
2002-03-19
期刊：
影响因子：
2.9
通讯作者：
Gennis, RB
中科院分区：
文献类型：
--
作者：
Barquera, B;Hellwig, P;Gennis, RB
来自霍乱弧菌的nqr操纵子在P-BAD启动子的调控下被克隆，该操纵子编码完整的六亚基、膜相关的、Na+易位的NADH：醌氧化还原酶（Na+-NQR）。该酶在霍乱弧菌中成功表达。为了促进这种钠泵酶的分子遗传学研究，构建了一种缺失nqr操纵子基因组拷贝的霍乱弧菌宿主菌株。通过使用在操纵子中的最后一个亚基NqrF亚基的羧基末端上含有六个组氨酸标签的载体，重组酶容易地通过亲和色谱法以高活性形式从洗涤剂溶解的霍乱弧菌膜中纯化。该重组酶在钠存在下具有高比活性。在每秒720个电子的周转数下评估NADH消耗。当使用十二烷基麦芽糖苷（DM）纯化时，分离的酶含有大约一种结合的泛醌，而如果使用洗涤剂LDAO代替，则分离的酶的醌含量可忽略不计。此外，用DM纯化的重组酶与O-2的反应速率相对较低（10-20 s（-1））。在稳态营业额，分离的重组酶表现出高达5倍的刺激钠和功能作为一个主要的钠泵，如先前报道的Na+-NQR从其他细菌来源。当重组到脂质体中时，重组Na+-NQR产生钠梯度和跨膜Δ Psi。SDS-PAGE解析了所有六个亚基，其中两个，NqrB和NqrC，含有共价结合的黄素。通过紫外-可见光谱监测的酶的氧化还原滴定，揭示了三个n = 2的氧化还原中心和一个n = 1的氧化还原中心，其中三个黄素和2Fe-2S中心的存在可以解释。霍乱弧菌Na+-NQR非常适合于结构研究和分子遗传学技术的使用，以解决NADH氧化与Na+跨膜泵送耦合的机制。
The nqr operon from Vibrio cholerae, encoding the entire six-subunit, membrane-associated, Na+-translocating NADH:quinone oxidoreductase (Na+-NQR), was cloned under the regulation of the P-BAD promoter. The enzyme was successfully expressed in V. cholerae. To facilitate molecular genetics studies of this sodium-pumping enzyme, a host strain of V. cholerae was constructed in which the genomic copy of the nqr operon was deleted. By using a vector containing a six-histidine tag on the carboxy terminus of the NqrF subunit, the last subunit in the operon, the recombinant enzyme was readily purified by affinity chromatography in a highly active form from detergent-solubilized membranes of V. cholerae. The recombinant enzyme has a high specific activity in the presence of sodium. NADH consumption was assessed at a turnover number of 720 electrons per second. When purified using dodecyl maltoside (DM), the isolated enzyme contains approximately one bound ubiquinone, whereas if the detergent LDAO is used instead, the quinone content of the isolated enzyme is negligible. Furthermore, the recombinant enzyme, purified with DM, has a relatively low rate of reaction with O-2 (10-20 s(-1)). In steady state turnover, the isolated, recombinant enzyme exhibits up to 5-fold stimulation by sodium and functions as a primary sodium pump, as reported previously for Na+-NQR from other bacterial sources. When reconstituted into liposomes, the recombinant Na+-NQR generates a sodium gradient and a Deltapsi across the membrane. SDS-PAGE resolves all six subunits, two of which, NqrB and NqrC, contain covalently bound flavin. A redox titration of the enzyme, monitored by UV-visible spectroscopy, reveals three n = 2 redox centers and one n = 1 redox center, for which the presence of three flavins and a 2Fe-2S center can account. The V. cholerae Na+-NQR is well-suited for structural studies and for the use of molecular genetics techniques in addressing the mechanism by which NADH oxidation is coupled to the pumping of Na+ across the membrane.