Dubourdieu M, DeMoss J A
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77030.
J Bacteriol. 1992 Feb;174(3):867-72. doi: 10.1128/jb.174.3.867-872.1992.
Respiratory nitrate reductase purified from the cell membrane of Escherichia coli is composed of three subunits, alpha, beta, and gamma, which are encoded, respectively, by the narG, narH, and narI genes of the narGHJI operon. The product of the narJ gene was deduced previously to be a highly charged, acidic protein which was not found to be associated with any of the purified preparations of the enzyme and which, in studies with putative narJ mutants, did not appear to be absolutely required for formation of the membrane-bound enzyme. To test this latter hypothesis, the narJ gene was disrupted in a plasmid which contained the complete narGHJI operon, and the operon was expressed in a narG::Tn10 insertion mutant. The chromosomal copy of the narJ gene of a wild-type strain was also replaced by the disrupted narJ gene. In both cases, when nar operon expression was induced, the alpha and beta subunits accumulated in a form which expressed only very low activity with either reduced methyl viologen (MVH) or formate as electron donors, although an alpha-beta complex separated from the gamma subunit is known to catalyze full MVH-linked activity but not the formate-linked activity associated with the membrane-bound complex. The low-activity forms of the alpha and beta subunits also accumulated in the absence of the NarJ protein when the gamma subunit (NarI) was provided from a multicopy plasmid, indicating that NarJ is essential for the formation of the active, membrane-bound complex. When both NarJ and NarI were provided from a plasmid in the narJ mutant, fully active, membrane-bound activity was formed. When NarJ only was provided from a plasmid in the narJ mutant, a cytosolic form of the alpha and beta subunits, which expressed significantly increased levels of the MVH-dependent activity, accumulated, and the alpha subunit appeared to be protected from the proteolytic clipping which occurred in the absence of NarJ. We conclude that NarJ is indispensible for the biogenesis of membrane-bound nitrate reductase and is involved either in the maturation of a soluble, active alpha-beta complex or in facilitating the interaction of the complex with the membrane-bound gamma subunit.
从大肠杆菌细胞膜中纯化得到的呼吸硝酸盐还原酶由三个亚基α、β和γ组成，它们分别由narGHJI操纵子的narG、narH和narI基因编码。先前推断narJ基因的产物是一种带电量高的酸性蛋白质，在任何纯化的酶制剂中均未发现其存在，并且在对假定的narJ突变体的研究中，它似乎并非膜结合酶形成所绝对必需的。为了验证后一个假设，在一个含有完整narGHJI操纵子的质粒中破坏narJ基因，并在narG::Tn10插入突变体中表达该操纵子。野生型菌株narJ基因的染色体拷贝也被破坏的narJ基因所取代。在这两种情况下，当诱导nar操纵子表达时，α和β亚基以一种形式积累，该形式以还原型甲基紫精（MVH）或甲酸盐作为电子供体时仅表现出非常低的活性，尽管已知从γ亚基分离出的α-β复合物可催化与MVH完全相关的活性，但不能催化与膜结合复合物相关的甲酸盐相关活性。当从多拷贝质粒提供γ亚基（NarI）时，α和β亚基的低活性形式在没有NarJ蛋白的情况下也会积累，这表明NarJ对于活性膜结合复合物的形成至关重要。当在narJ突变体中从质粒同时提供NarJ和NarI时，形成了完全活性的膜结合活性。当仅在narJ突变体中从质粒提供NarJ时，积累了α和β亚基的胞质形式，其表达的MVH依赖性活性水平显著增加，并且α亚基似乎受到保护，免受在没有NarJ的情况下发生的蛋白水解剪切。我们得出结论，NarJ对于膜结合硝酸盐还原酶的生物合成是不可或缺的，并且参与可溶性活性α-β复合物的成熟或促进该复合物与膜结合γ亚基的相互作用。
