STRUCTURE AND MECHANISM OF THE E COLI MANNITOL PERMEASE

大肠杆菌甘露醇渗透酶的结构和机制

• 批准号: 2175139
• 负责人: GARY R JACOBSON
• 金额: $ 21.34万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-07-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2175139
• 关键词: Escherichia coli; antibody; carbohydrate receptor; carbohydrate transport; crystallization; enzyme mechanism; enzyme structure; gene complementation; laboratory rabbit; mutant; nucleic acid sequence; permease; phosphorylation; polymerase chain reaction; protein structure function

Our laboratory is investigating the structure and mechanism of the enzyme/permease responsible for the coupled transport and phosphorylation of the hexitol D-mannitol in Escherichia coli. This protein is an integral membrane component of the bacterial phosphotransferase system (PTS) and also serves as a chemotactic receptor for D-mannitol in E, coli. Over the past decade, its structure and catalytic mechanism have been extensively studied by biochemical, biophysical and genetic techniques. The mannitol permease remains the most attractive transport protein of the PTS to study because it has been purified, its gene has been cloned and sequenced, and its overall disposition with respect to the membrane is known. The broad objective of these studies is to determine in as much molecular detail as possible the mechanism by which the mannitol permease carries out its receptor and transport functions. This is not completely known for any membrane transport protein, and the results of these studies should therefore add fundamental knowledge to the understanding of transport protein structure and function. Since all cells rely on proper functioning of transport proteins for interaction with their environment, and some diseased cells may lack one or more of these functions, the study of these fundamental processes is essential in understanding the basic principles underlying growth and metabolism in both normal and diseased cells. Moreover, since many pathogenic bacteria have a PTS, but this system is absent in eucaryotes, it is a potential target for antibacterial agents. In the proposed project continuation, the aims are to: 1) continue to isolate and characterize mutants in the mannitol permease; 2) use complementation studies and the isolation of pseudorevertants to characterize important interactions in the permease; 3) study the structure of the permease including attempts to crystallize it; and 4) study a structurally related protein, ORF162, which may be a regulatory link between the PTS and nitrogen assimilation in bacteria. A combination of biochemical, biophysical, molecular biological and genetic techniques will be used to accomplish these aims. Collaborations and consultations with other workers in this area will ensure a complementary, rather than a competitive expenditure of time and effort in this project. Successful completion of this work will lead to significant progress toward our overall goal of understanding the molecular basis of transport and chemoreception in this system.

我们的实验室正在研究的结构和机制， 负责偶联转运和磷酸化的酶/通透酶 己糖醇D-甘露醇在大肠杆菌。 这种蛋白质是 细菌磷酸转移酶系统的整体膜组件 (PTS)并且还作为E中D-甘露醇的趋化性受体， 杆菌 在过去的十年里，它的结构和催化机制， 生物化学、生物物理学和遗传学已经广泛地研究了 技术. 甘露醇通透酶仍然是最有吸引力的转运蛋白 PTS的蛋白质进行研究，因为它已经被纯化，其基因具有 克隆和测序，以及其在 膜是已知的。 这些研究的主要目标是 尽可能多地确定分子细节， 甘露醇通透酶执行其受体和转运功能。 这对于任何膜转运蛋白都不是完全已知的， 因此，这些研究的结果应该增加基础知识， 对转运蛋白结构和功能的理解。 由于所有 细胞依赖于转运蛋白的正常功能进行相互作用 与其环境，和一些病变细胞可能缺乏一个或多个 这些功能，这些基本过程的研究是必要的 了解生长和新陈代谢的基本原理 在正常细胞和病变细胞中。 此外，由于许多病原体 细菌有一个PTS，但这个系统在真核生物中是不存在的，它是一个 抗菌剂的潜在靶点。 拟建工程 继续，目标是：1）继续分离和表征 甘露醇渗透酶的突变体; 2）使用互补研究， 分离假回复突变体以表征 3）研究了通透酶的结构，包括尝试 使其结晶;以及4）研究结构相关的蛋白质，ORF 162， 这可能是PTS和氮同化之间的调节联系 在细菌中。 结合生物化学，生物物理，分子 将利用生物和遗传技术来实现这些目标。 与该领域其他工作者的合作和协商将 确保时间的消耗是互补的，而不是竞争性的， 在这个项目上的努力。 这项工作的顺利完成将导致 我们的总体目标是了解 在这个系统中的运输和化学感受的分子基础。