2-oxoglutarate and the PII homologues NifI1 and NifI2 regulate nitrogenase activity in cell extracts of Methanococcus maripaludis
2-oxoglutarate and the PII homologues NifI1 and NifI2 regulate nitrogenase activity in cell extracts of Methanococcus maripaludis
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DOI：
10.1111/j.1365-2958.2005.04621.x
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发表时间：
2005-06-01
影响因子：
3.6
通讯作者：
Leigh, JA
中科院分区：
文献类型：
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作者：
Dodsworth, JA;Cady, NC;Leigh, JA
产甲烷古菌maripaludis产甲烷球菌（Methanococcus maripaludis）中固氮或关闭的翻译后调控不像某些细菌那样涉及可检测到的二氮酶还原酶的共价修饰，并且编码PII同源物Nifl(1)和Nifl(2)的基因都是必需的，这表明了一种新的机制。为了进一步了解关闭的机制，我们检测了野生型和nifI突变株的细胞提取物在缺乏或存在氮状态潜在信号的情况下的氮酶活性。Delta nifl 1和nifl 2菌株提取物的活性是野生型细胞提取物的6倍。在野生型提取物中添加2-氧戊二酸酯可使活性提高5倍，其水平与Delta nifl(1) nifl(2)提取物相似。2-氧戊二酸不影响Delta nifl 1、nifl 2或单一nifl突变体提取物的活性。此外，基因互补的nifI突变体的提取物恢复了野生型特征，表明体外效应与体内效应相关。2-氧葡萄糖酸酯的提取和定量表明，固氮细胞的浓度比关闭和氨培养细胞高10倍。我们提出了一个关闭模型，其中Nifl蛋白对氮酶活性具有抑制作用，这种抑制作用被高水平的2-氧戊二酸抵消，后者作为氮限制的信号。
Post-translational regulation of nitrogen fixation, or switch-off, in the methanogenic archaeon Methanococcus maripaludis does not involve detectable covalent modification of the dinitrogenase reductase as in some bacteria, and the genes encoding the PII homologues Nifl(1) and Nifl(2) are both required, indicating a novel mechanism. To further understand the mechanism of switch-off, we assayed nitrogenase activity in cell extracts from wild-type and nifI mutant strains in the absence or presence of potential signals of nitrogen status. Activity in extracts from a Delta nifl 1 nifl 2 strain was sixfold higher than in extracts from wild-type cells. Addition of 2-oxoglutarate to wild-type extracts enhanced activity up to fivefold, a level similar to that observed in Delta nifl(1) nifl(2) extracts. 2-Oxoglutarate did not affect activity in Delta nifl 1 nifl 2 or single nifl mutant extracts. Furthermore, extracts from genetically complimented nifI mutants regained wild-type characteristics, indicating an in vitro correlation with in vivo effects. Extraction and quantification of 2-oxoglutarate indicated concentrations 10-fold higher in nitrogenfixing cells than in switched-off and ammonium-grown cells. We propose a model for switch-off where the Nifl proteins have an inhibitory effect on nitrogenase activity that is counteracted by high levels of 2- oxoglutarate, which acts as a signal of nitrogen limitation.