NAD+ biosynthesis in bacteria is controlled by global carbon/nitrogen levels via PII signaling
NAD+ biosynthesis in bacteria is controlled by global carbon/nitrogen levels via PII signaling
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DOI：
10.1074/jbc.ra120.012793
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发表时间：
2020-03
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通讯作者：
A. Santos;E. C. Gerhardt;Erick Parize;F. O. Pedrosa;M. Steffens;L. S. Chubatsu;E. M. Souza;L. Passaglia;F. H. Sant’Anna;Gustavo A de Souza;L. Huergo;K. Forchhammer
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文献类型：
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作者：
A. Santos;E. C. Gerhardt;Erick Parize;F. O. Pedrosa;M. Steffens;L. S. Chubatsu;E. M. Souza;L. Passaglia;F. H. Sant’Anna;Gustavo A de Souza;L. Huergo;K. Forchhammer
NAD+是参与核心代谢氧化还原反应的中心代谢物。原核NAD合成酶NadE催化NAD+生物合成的最后一步，将烟酸腺嘌呤二核苷酸（NaAD）转化为NAD+。NadE家族的一些成员使用l-谷氨酰胺作为氮供体，并被命名为NadEGln。以前的基因邻域分析表明，细菌nadE基因经常与编码调节信号转导蛋白PII的基因聚簇，这表明这些蛋白质之间的功能关系，在响应营养状态和细菌细胞的碳/氮比。在这里，使用亲和层析，生物信息学分析，NAD合成酶活性，和生物层干涉测定，我们表明，PII和NadEGln物理相互作用在体外，这种复杂的解除NadEGln负反馈抑制NAD+。这种机制在远亲细菌中是保守的。值得注意的是，PII蛋白变构效应物和细胞氮水平指示剂2-酮戊二酸（2-OG）在生理范围内抑制PII-NadEGln复合物的形成。这些结果表明ATP、ADP、2-OG、PII-感应的谷氨酰胺和NAD+的水平之间的相互作用，代表可以平衡核心氮和碳代谢物水平的代谢中心。我们的研究结果支持的概念，PII蛋白作为一个可分离的调节亚基NadEGln，从而使NAD+生物合成的控制根据细菌细胞的营养状况。
NAD+ is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+. Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadEGln. Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+. This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell.