Data_Sheet_1_The PII protein interacts with the Amt ammonium transport and modulates nitrate/nitrite assimilation in mycobacteria.pdf

PII proteins are signal transduction proteins that belong to a widely distributed family of proteins involved in the modulation of different metabolisms in bacteria. These proteins are homotrimers carrying a flexible loop, named T-loop, which changes its conformation due to the recognition of diverse key metabolites, ADP, ATP, and 2-oxoglutarate. PII proteins interact with different partners to primarily regulate a set of nitrogen pathways. In some organisms, PII proteins can also control carbon metabolism by interacting with the biotin carboxyl carrier protein (BCCP), a key component of the acetyl-CoA carboxylase (ACC) enzyme complex, inhibiting its activity with the consequent reduction of fatty acid biosynthesis. Most bacteria contain at least two PII proteins, named GlnB and GlnK, with different regulatory roles. In mycobacteria, only one PII protein was identified, and the three-dimensional structure was solved, however, its physiological role is unknown. In this study we purified the Mycobacterium tuberculosis (M. tb) PII protein, named GlnB, and showed that it weakly interacts with the AccA3 protein, the α subunit shared by the three different, and essential, Acyl-CoA carboxylase complexes (ACCase 4, 5, and 6) present in M. tb. A M. smegmatis deletion mutant, ∆MsPII, exhibited a growth deficiency on nitrate and nitrite as unique nitrogen sources, and accumulated nitrite in the culture supernatant. In addition, M. tb PII protein was able to interact with the C-terminal domain of the ammonium transporter Amt establishing the ancestral role for this PII protein as a GlnK functioning protein.

PII蛋白是一类广泛分布于细菌中，参与调节多种代谢活动的信号转导蛋白。此类蛋白为同源三聚体，携带一个具有柔性的T环结构域，该结构域因其对多种关键代谢物（如ADP、ATP和2-氧戊二酸）的识别而改变其构象。PII蛋白通过与不同的合作伙伴相互作用，主要调节一系列氮代谢途径。在某些生物体中，PII蛋白还能通过与生物素羧基载体蛋白（BCCP）相互作用来控制碳代谢，BCCP是乙酰辅酶A羧化酶（ACC）酶复合体的重要组成部分，PII蛋白通过与BCCP的相互作用抑制其活性，从而降低脂肪酸的生物合成。大多数细菌至少含有两种PII蛋白，分别为GlnB和GlnK，它们在调控功能上有所不同。在分枝杆菌中，仅鉴定出一种PII蛋白，并确定了其三维结构，但其生理功能尚不明确。在本研究中，我们纯化了结核分枝杆菌（M. tb）的PII蛋白，命名为GlnB，并证实其与AccA3蛋白存在微弱的相互作用，AccA3蛋白是M. tb中三种不同且必需的酰基辅酶A羧化酶复合体（ACCase 4、5和6）共有的α亚基。一个恶臭假单胞菌的缺失突变体∆MsPII在以硝酸和亚硝酸为唯一氮源时表现出生长缺陷，并在培养基上清中积累亚硝酸。此外，M. tb的PII蛋白能够与铵转运蛋白Amt的C端结构域相互作用，确立了该PII蛋白作为GlnK功能蛋白的祖先角色。