结核分枝杆菌由繁琐的信号传导和蛋白质网络组成,参与细菌的生存并增强其发病机理。分枝杆菌PhoH2(Mt-PhoH2)是信号元件和预测性磷酸盐饥饿蛋白,其以ATP依赖性方式起作用。这里,我们通过生物物理阐述了Mt-PhoH2的表征,生物化学,和计算方法。除了其固有的ATP酶活性,生化实验揭示了其GTP酶活性,并且两种活性都依赖于金属离子。镁,锰,铜,铁,镍,锌,铯,钙,检查锂对活性的影响,用10mM的Mg2离子发现了最佳活性。观察到3µMt-PhoH2的动力学参数为Km4.873±0.44µM,Vmax12.3817±0.084µM/min/mg,Kcat0.0075±0.00005s-1,和Kcat/Km0.0015±0.000001µM-1s-1和GTP。在GTP作为底物的情况下,观察到Mt-PhoH2的酶活性降低20%,结合亲和力增加50%。底物ADP和GDP抑制Mt-PhoH2的ATP酶和GTP酶活性。CD光谱显示了Mt-PhoH2二级结构中α螺旋的优势,并且该结构模式在添加ATP和GTP时发生了改变。计算机抑制剂筛选显示ML141和NAV_2729是Mt-PhoH2催化活性的两种潜在抑制剂。Mt-PhoH2是分枝杆菌生长所必需的,因为其敲除菌株显示出降低的生长效应。总的来说,本文强调了Mt-PhoH2正常运作所必需的因素,Mt-PhoH2是毒素-抗毒素机制的参与者,也可能在磷酸盐饥饿中起重要作用。
Mycobacterium tuberculosis is composed of a cumbersome signaling and protein network which partakes in bacterial survival and augments its pathogenesis. Mycobacterial PhoH2 (Mt-PhoH2) is a signaling element and a predictive phosphate starvation protein that works in an ATP-dependent manner. Here, we elaborated the characterization of Mt-PhoH2 through biophysical, biochemical, and computational methods. In addition to its intrinsic ATPase activity, the biochemical experiments revealed its GTPase activity and both activities are metal ion dependent. Magnesium, manganese, copper, iron, nickel, zinc, cesium, calcium, and lithium were examined for their effect on activity, and the optimum activity was found with 10 mM of Mg2+ ions. The kinetic parameters of 3 µM Mt-PhoH2 were observed as Km 4.873 ± 0.44 µM, Vmax 12.3817 ± 0.084 µM/min/mg, Kcat 0.0075 ± 0.00005 s-1, and Kcat/Km 0.0015 ± 0.000001 µM-1 s-1 with
GTP. In the case of
GTP as a substrate, a 20% decrease in enzymatic activity and a 50% increase in binding affinity of Mt-PhoH2 were observed. The substrates ADP and GDP inhibit the ATPase and GTPase activity of Mt-PhoH2. CD spectroscopy showed the dominance of alpha helix in the secondary structure of Mt-PhoH2, and this structural pattern was altered upon addition of ATP and
GTP. In silico inhibitor screening revealed ML141 and NAV_2729 as two potential inhibitors of the catalytic activity of Mt-PhoH2. Mt-PhoH2 is essential for mycobacterial growth as its knockdown strain showed a decreased growth effect. Overall, the present article emphasizes the factors essential for the proper functioning of Mt-PhoH2 which is a participant in the toxin-antitoxin machinery and may also play an important role in phosphate starvation.