Williams S B, Stewart V
Section of Microbiology, Cornell University, Ithaca, NY 14853-8101, USA.
Mol Microbiol. 1997 Dec;26(5):911-25. doi: 10.1046/j.1365-2958.1997.6262002.x.
Anaerobic respiratory gene expression in Escherichia coli is differentially controlled by nitrate and nitrite through dual interacting two-component regulatory systems. The NarX sensor is one of two membrane-spanning sensor kinases that control the phosphorylation state of two DNA-binding response regulators. We have studied NarX autophosphorylation in crude membrane preparations from cells that overexpress NarX protein. The low basal autophosphorylation rate was stimulated about sixfold and threefold by nitrate and nitrite respectively. This demonstrates that nitrate and nitrite differentially activate NarX autokinase activity. We also isolated single-residue substitutions in NarX that affect its ability to respond to or discriminate between nitrate and nitrite. Most of these substitutions affect residues within the conserved P-box sequence in the periplasmic domain. We characterized several of the mutants in vivo, by monitoring ligand-regulated gene expression, and in vitro, by monitoring ligand-responsive autophosphorylation. At least one change, K491 (Lys at position 49 changed to Ile), resulted in a protein with greatly impaired ability to discriminate between nitrate and nitrite. Other changes (H45E and R59K) resulted in proteins that responded normally to nitrate but were unable to respond to nitrite. These results implicate the P-box region in discrimination between subtly different small molecules.
大肠杆菌中厌氧呼吸基因的表达通过双重相互作用的双组分调节系统受到硝酸盐和亚硝酸盐的差异控制。NarX传感器是控制两种DNA结合响应调节因子磷酸化状态的两种跨膜传感器激酶之一。我们研究了在过表达NarX蛋白的细胞的粗制膜制剂中的NarX自磷酸化。低基础自磷酸化速率分别被硝酸盐和亚硝酸盐刺激约6倍和3倍。这表明硝酸盐和亚硝酸盐差异激活NarX自激酶活性。我们还分离出NarX中的单残基取代，这些取代影响其对硝酸盐和亚硝酸盐的响应或区分能力。这些取代中的大多数影响周质结构域中保守的P盒序列内的残基。我们通过监测配体调节的基因表达在体内对几个突变体进行了表征，并通过监测配体响应的自磷酸化在体外进行了表征。至少有一个变化，K491（第49位的赖氨酸变为异亮氨酸），导致一种蛋白质区分硝酸盐和亚硝酸盐的能力大大受损。其他变化（H45E和R59K）导致蛋白质对硝酸盐正常反应但无法对亚硝酸盐反应。这些结果表明P盒区域在区分细微不同的小分子中起作用。
