NifL of Klebsiella pneumoniae carries an N-terminally bound FAD cofactor, which is not directly required for the inhibitory function of NifL.
NifL of Klebsiella pneumoniae carries an N-terminally bound FAD cofactor, which is not directly required for the inhibitory function of NifL.
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肺炎克雷伯菌的 NifL 携带 N 末端结合的 FAD 辅助因子，该因子不是 NifL 的抑制功能所直接需要的。
DOI：
10.1111/j.1574-6968.1997.tb12791.x
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发表时间：
1997
影响因子：
2.1
通讯作者：
R. Schmitz
中科院分区：
文献类型：
--
作者：
R. Schmitz
在肺炎克雷伯氏菌中，NifL在分子氧或结合氮存在下拮抗转录激活因子NifA的作用。为了确定哪些辅因子可能参与氧传感机制，我们纯化并分析了大肠杆菌麦芽糖结合蛋白，MalE和NifL之间的融合蛋白。在严格厌氧条件下合成和纯化的NifL不含显著量的铁或酸不稳定的硫，表明不存在氧敏感铁-硫簇。然而，以其抑制形式纯化的NifL蛋白含有0.3 +/- 0.01 mol FAD和小于0.01 mol FMN/mol NifL，表明存在FAD作为辅因子。在不存在氧和结合氮的情况下合成的NifL的表征表明，NifL的非抑制形式也含有FAD（0.54 mol FAD/mol NifL）。使用MalE和NifL的不同部分之间的融合，我们将FAD的结合位点定位于NifL的N-末端结构域。这些结果和我们先前观察到的NifL的C-末端结构域足以抑制NifA活性表明N-末端结合的FAD不是NifL的抑制活性直接需要的。这一观察结果得到以下发现的支持：纯化的NifL脱辅基蛋白在体外仍然能够抑制NifA的转录激活。
In Klebsiella pneumoniae NifL antagonizes the action of the transcriptional activator NifA in the presence of molecular oxygen or combined nitrogen. To determine what cofactors might be involved in the oxygen sensing mechanism, we purified and analyzed fusion proteins made between the Escherichia coli maltose binding protein, MalE, and NifL. NifL synthesized and purified under strictly anaerobic conditions did not contain significant amounts of iron or acid-labile sulfur indicating the absence of an oxygen sensing iron-sulfur cluster. However, NifL protein purified in its inhibitory form contained 0.3 +/- 0.01 mol FAD and less than 0.01 mol FMN per mol NifL suggesting the presence of FAD as a cofactor. Characterization of NifL synthesized in the absence of oxygen and combined nitrogen showed that the non-inhibitory form of NifL also contained FAD (0.54 mol FAD per mol NifL). Using fusions between MalE and different portions of NifL we localized the binding site of FAD to the N-terminal domain of NifL. These results and our previous observation that the C-terminal domain of NifL is sufficient to inhibit NifA activity indicate that the N-terminally bound FAD is not directly required for the inhibitory activity of NifL. This observation is supported by the finding that purified apoprotein of NifL was still able to inhibit transcriptional activation by NifA in vitro.
DOI：
10.1073/pnas.94.20.10541
发表时间：
1997-09-30
影响因子：
11.1
作者：
Rebbapragada, A;Johnson, MS;Taylor, BL
通讯作者：
Taylor, BL
最大化含有 lambda PL 启动子的质粒载体的基因表达：过量产生转录终止因子 rho 的策略。
DOI：
10.1073/pnas.82.1.88
发表时间：
1985
影响因子：
11.1
作者：
Mott,JE;Grant,RA;Ho,YS;Platt,T
通讯作者：
Platt,T