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

大肠杆菌氨气通道AmtB的作用机制

• 批准号: 8207219
• 负责人: Joanne Hsu
• 金额: $ 1.62万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8207219
• 关键词: Active Sites; Address; Affect; Amino Acids; Ammonia; Ammonium; Antigens; Assimilations; Bacteria; Binding; Binding Sites; Biological; Carbon Dioxide; Carrier Proteins; Cell membrane; Cell physiology; Cells; Chlamydomonas reinhardtii; Coupled; Defect; Disease; Enterobacteriaceae; Enzymes; Erythrocytes; Escherichia coli; Functional disorder; Gases; Genes; Glutamate-Ammonia Ligase; Glutamine; Green Algae; Growth; Human; Macaca mulatta; Metabolic; Molecular Biology; Molecular Genetics; Mutation; Names; Organism; Phospholipids; Physiological; Protein Region; Proteins; Salmonella typhimurium; Structural Genes; Suppressor Mutations; Testing; Work; base; human disease; loss of function; protein misfolding; uptake

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.

一、项目概要： 氨转运 (Amt) 蛋白和恒河猴 (Rh) 蛋白是仅有的两种已识别的生物气体 生物体之间的通道。 Rh 蛋白是最为人所知的人类红细胞抗原， CO2 气体通道。它们是绿藻莱茵衣藻最佳生长所必需的 在高CO2下。 Amt 蛋白被称为 Rh 蛋白的祖先同源物，是气体通道 氨。它们是肠道细菌大肠杆菌和鼠伤寒沙门氏菌最佳生长所必需的 在低 NH3 条件下。由于细胞膜的磷脂双层可渗透气体种类，原因 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 的 NH3 转运似乎与谷氨酰胺合成酶偶联 它将 NH3 同化成谷氨酰胺。因此，在本提案中，我们试图了解 AmtB 在 E. 大肠杆菌与谷氨酰胺合成酶功能性偶联，并推测 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 孔传递到谷氨酰胺合成酶的活性位点，从而提高速率 NH3 的同化。该提案的两个具体目标之一是鉴定氨基酸突变 AmtB 在低 NH3 条件下会损害生长和铵吸收活性。这些 amtB 突变不应该 导致 AmtB 整体蛋白质错误折叠或 AmtB 孔功能丧失，但局部影响 蛋白质的细胞质区域是谷氨酰胺合成酶的结合位点。第二个具体目标 目的是使用分子生物学和遗传学方法测试 AmtB 和 GS 之间的关联。 二.关联： 这项针对大肠杆菌 AmtB 的拟议研究将使我们能够更好地了解气体通道的工作原理 帮助维持健康的细胞生理机能。了解气道功能将有助于我们确定气道的基础 人类疾病中的功能障碍，例如 Rh 缺失病。