Mechanism of action of the ammonia gas channel AmtB in E. coli

一、项目总结： 氨运输(AMT)蛋白和恒河猴(Rh)蛋白是仅有的两种已鉴定的生物气体 有机体之间的通道。Rh蛋白最为人所知的是人类红细胞上的抗原， 二氧化碳的气体通道。它们是绿藻衣藻最佳生长所必需的。 二氧化碳含量很高。AMT蛋白被称为Rh蛋白的-祖先同源蛋白，是 NH3。它们是肠杆菌、大肠杆菌和鼠伤寒沙门氏菌最佳生长所必需的。 在低氨氮条件下。由于细胞膜的磷脂双层对气体物种是渗透的，所以 为什么细胞需要气体通道尚不清楚。大肠杆菌和鼠伤寒沙门氏菌的生理学研究 表明在低NH3浓度下，AmtB转运NH3似乎与谷氨酰胺合成酶偶联 将NH3同化为谷氨酰胺。因此，在这个提案中，我们试图理解AmtB是如何在E. Coli在功能上与谷氨酰胺合成酶偶联，并假设AmtB和谷氨酰胺合成酶 身体上的联系。AmtB和谷氨酰胺合成酶之间的物理接触可能允许直接 将NH3从AmtB的孔道输送到谷氨酰胺合成酶的活性部位，从而提高了合成速率 对NH3的同化作用。这项提议的两个具体目标之一是确定氨基酸突变 在低氨氮条件下，AmtB对生长和氨吸收活性有不利影响。这些amtB突变不应该 导致AmtB的整体蛋白质错误折叠或AmtB的孔功能丧失，但更多是局部影响 蛋白质的细胞质区域，是其与谷氨酰胺合成酶的结合部位。第二个具体目标 是利用分子生物学和遗传学的方法来测试AmtB和GS之间的联系。 二、相关性： 这项对大肠杆菌AmtB的拟议研究将使我们能够更好地了解气体通道是如何工作的 以帮助维持健康的细胞生理。了解气体通道的功能将有助于我们确定 人类疾病中的功能障碍，如Rh缺陷症。

I. Project Summary: Ammonia transport (Amt) proteins and Rhesus (Rh) proteins are the only two identified biological gas channels among organisms. Rh proteins, which are best known as antigens on human red blood cells, are gas channels for CO2. They are required for optimal growth of the green alga Chlamydomonas reinhardtii at high C02. Amt proteins, which are known as the-ancestralhomolog to Rh proteins, are gas channels for NH3. They are required for optimal growth of enteric bacteria Escherichia coli and Salmonella typhimurium at low NH3. Since phospholipid bilayers of the cell membrane are permeable to the gas species, the reason why gas channels are needed by cells is not known. Physiological studies in E. coli and S. typhimurium show that at low NH3 concentrations NH3 transport by AmtB appears to be coupled to glutamine synthetase which assimilates NH3 into glutamine. Therefore, in this proposal we seek to understand how AmtB in E. coli is functionally coupled to glutamine synthetase and hypothesize that AmtB and glutamine synthetase associate physically. The physical contact between AmtB and glutamine synthetase may allow the direct delivery of NH3 from the pore of AmtB to the active site of glutamine synthetase and hence increase the rate of assimilation of NH3. One of the two specific aims of this proposal is to identify amino acid mutations in AmtB that impair growth and ammonium uptake activity at low NH3. These amtB mutations should not cause global protein misfolding of AmtB or loss of function of the pore of AmtB but rather locally affect cytoplasmic regions of the protein that are its binding site for glutamine synthetase. The second specific aim is to test the association between AmtB and GS using molecular biology and genetics approaches. II. Relevance: This proposed study of E. coli AmtB will enable us to gain a better understanding of how gas channels work to help maintain healthy cell physiology. Knowing gas channel function will help us identify the basis of dysfunction in human disease such as Rh null disease.