Money T, Jones T, Dixon R, Austin S
Nitrogen Fixation Laboratory, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, Norfolk, United Kingdom.
J Bacteriol. 1999 Aug;181(15):4461-8. doi: 10.1128/JB.181.15.4461-4468.1999.
In Azotobacter vinelandii, activation of nif gene expression by the transcriptional regulatory enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo. NIFL is a novel redox-sensing flavoprotein which is also responsive to adenosine nucleotides in vitro. Inhibition of NIFA activity by NIFL requires stoichiometric amounts of the two proteins, implying that the mechanism of inhibition is by direct protein-protein interaction rather than by catalytic modification of the NIFA protein. The formation of the inhibitory complex between NIFL and NIFA may be regulated by the intracellular ATP/ADP ratio. We show that adenosine nucleotides promote complex formation between purified NIFA and NIFL in vitro, allowing isolation of the NIFL-NIFA complex. The complex can also be isolated from cell extracts containing coexpressed NIFL and NIFA in the presence of MgADP. Removal of the nucleotide causes dissociation of the complex. Experiments with truncated proteins demonstrate that the amino-terminal domain of NIFA and the C-terminal region of NIFL potentiate the ADP-dependent stimulation of NIFL-NIFA complex formation.
在棕色固氮菌中，转录调控增强子结合蛋白NIFA对固氮基因表达的激活作用，受传感蛋白NIFL的控制，以响应体内氧气和固定氮水平的变化。NIFL是一种新型的氧化还原感应黄素蛋白，在体外也对腺苷核苷酸有反应。NIFL对NIFA活性的抑制需要化学计量的两种蛋白质，这意味着抑制机制是通过直接的蛋白质-蛋白质相互作用，而不是通过对NIFA蛋白的催化修饰。NIFL和NIFA之间抑制性复合物的形成可能受细胞内ATP/ADP比值的调节。我们发现，腺苷核苷酸在体外促进纯化的NIFA和NIFL之间形成复合物，从而能够分离出NIFL-NIFA复合物。在MgADP存在的情况下，该复合物也可以从共表达NIFL和NIFA的细胞提取物中分离出来。去除核苷酸会导致复合物解离。对截短蛋白的实验表明，NIFA的氨基末端结构域和NIFL的羧基末端区域增强了ADP对NIFL-NIFA复合物形成的刺激作用。
