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: 2175140
• 负责人: GARY R JACOBSON
• 金额: $ 23.57万
• 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/2175140
• 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.

我们实验室正在研究其结构和机制 负责耦合转运和磷酸化的酶/通透性 大肠杆菌中的己糖醇 D-甘露醇。 这种蛋白质是一种 细菌磷酸转移酶系统的完整膜成分 (PTS)，也作为 E 中 D-甘露醇的趋化受体， 大肠杆菌。 近十年来，其结构和催化机制得到了不断的发展。 经过生物化学、生物物理和遗传学的广泛研究 技术。 甘露醇渗透酶仍然是最具吸引力的运输工具 要研究的 PTS 蛋白，因为它已被纯化，其基因已 已被克隆和测序，以及它的整体处置 该膜是已知的。 这些研究的总体目标是 尽可能详细地确定分子机制 甘露醇渗透酶发挥其受体和转运功能。 对于任何膜转运蛋白来说，这一点尚不完全清楚，并且 因此，这些研究的结果应该增加基础知识 了解转运蛋白的结构和功能。 既然所有 细胞依赖转运蛋白的正常功能进行相互作用 与其环境有关，一些患病细胞可能缺乏一种或多种 这些功能，对这些基本过程的研究至关重要 了解生长和新陈代谢的基本原理 在正常细胞和患病细胞中。 此外，由于许多致病 细菌有PTS，但真核生物没有这个系统，它是 抗菌剂的潜在靶点。 在拟议的项目中 继续，目标是：1）继续分离和表征 甘露醇通透酶突变体； 2）利用互补研究和 分离假回复体以表征重要的相互作用 渗透； 3）研究渗透酶的结构，包括尝试 使其结晶； 4) 研究结构相关的蛋白质 ORF162， 这可能是 PTS 和氮同化之间的调节联系 在细菌中。 生物化学、生物物理学、分子学的结合 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 确保时间和成本的支出是互补的，而不是竞争的 在这个项目中的努力。 这项工作的成功完成将导致 我们在理解这一总体目标方面取得了重大进展 该系统中运输和化学感受的分子基础。