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: 8010195
• 负责人: Joanne Hsu
• 金额: $ 3.07万
• 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/8010195
• 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; Homologous Gene; 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

DESCRIPTION (provided by applicant): 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-ancestral homolog 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.

描述（由申请人提供）：I。项目摘要：氨转运（AMT）蛋白质和恒河猴（RH）蛋白是生物体中仅有的两个被鉴定的生物气通道。 RH蛋白质是人类红细胞上最著名的抗原，是CO2的气通道。它们是在高C02处最佳生长的最佳生长。 AMT蛋白被称为rh蛋白的官能同源物，是NH3的气通道。它们是肠细菌大肠杆菌和鼠伤寒沙门氏菌在低NH3处的最佳生长所必需的。由于细胞膜的磷脂双层可渗透到气体物种上，因此尚不清楚细胞所需的气通道。大肠杆菌和鼠伤寒链球菌的生理研究表明，在低NH3浓度下，AMTB的NH3转运似乎与谷氨酰胺合成酶偶联，将NH3吸收到谷氨酰胺中。因此，在此提案中，我们试图了解大肠杆菌中的AMTB如何在功能上耦合到谷氨酰胺合成酶，并假设AMTB和谷氨酰胺合成酶在身体上均匀。 AMTB和谷氨酰胺合成酶之间的物理接触可以使NH3从AMTB的孔直接递送到谷氨酰胺合成酶的活性位点，从而增加NH3的同化速率。该提案的两个具体目的之一是鉴定AMTB中氨基酸突变，这些氨基酸突变会损害NH3低的生长和铵摄取活性。这些AMTB突变不应引起AMTB的全局蛋白质折叠或AMTB孔隙功能丧失，而是局部影响蛋白质的细胞质区域，这是其是谷氨酰胺合成酶的结合位点。第二个具体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。 ii。相关性：对大肠杆菌AMTB的这项拟议的研究将使我们能够更好地了解天然气通道如何帮助维持健康的细胞生理。了解气体通道功能将有助于我们确定人类疾病（例如RH无效疾病）中功能障碍的基础。