NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli
NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli
复制标题
DOI：
10.1046/j.1365-2958.1998.00795.x
复制
发表时间：
1998-05-01
影响因子：
3.6
通讯作者：
Giordano, G
中科院分区：
文献类型：
--
作者：
Blasco, F;Dos Santos, JP;Giordano, G
大肠杆菌中活性膜结合硝酸还原酶A的形成需要存在三个亚基，NarG，NarH和NarI，以及第四种蛋白质NarJ，其不是活性硝酸还原酶的一部分。在narJ菌株中，NarG和NarH亚基都与不稳定和无活性的NarGH复合物相关。在将纯化的NarJ-6 His多肽加入到narJ菌株的细胞上清液中后，在体外观察到该复合物的显著活化。一旦narJ突变体的ape-酶NarGHI通过NarI亚基锚定到膜上，它就不能在体外被NarJ再活化。NarJ蛋白特异性地识别催化NarG亚基，荧光、电子顺磁共振（EPR）光谱和基于电感耦合等离子体发射光谱（ICPES）的钼定量清楚地表明，在不存在NarJ的情况下，NarGH复合物中不存在钼辅因子。我们建议，NarJ是一个特定的伴侣，结合到NarG，因此可能会保持它在一个适当的竞争力开放构象的钼辅因子插入发生，导致催化活性酶。在钼辅因子插入到猿-硝酸还原酶中后，NarJ然后从活化的酶解离。
The formation of active membrane-bound nitrate reductase A in Escherichia coli requires the presence of three subunits, NarG, NarH and NarI, as well as a fourth protein, NarJ, that is not part of the active nitrate reductase. In narJ strains, both NarG and NarH subunits are associated in an unstable and inactive NarGH complex. A significant activation of this complex was observed in vitro after adding purified NarJ-6His polypeptide to the cell supernatant of a narJ strain, Once the ape-enzyme NarGHI of a narJ mutant has become anchored to the membrane via the NarI subunit, it cannot be reactivated by NarJ in vitro. NarJ protein specifically recognizes the catalytic NarG subunit, Fluorescence, electron paramagnetic resonance (EPR) spectroscopy and molybdenum quantification based on inductively coupled plasma emission spectroscopy (ICPES) clearly indicate that, in the absence of NarJ, no molybdenum cofactor is present in the NarGH complex. We propose that NarJ is a specific chaperone that binds to NarG and may thus keep it in an appropriate competent-open conformation for the molybdenum cofactor insertion to occur, resulting in a catalytically active enzyme. Upon insertion of the molybdenum cofactor into the ape-nitrate reductase, NarJ is then dissociated from the activated enzyme.