Transport and Metabolism of Folate Analogs in E. coli

叶酸类似物在大肠杆菌中的转运和代谢

• 批准号: 6754128
• 负责人: JACALYN M GREEN
• 金额: $ 21.78万
• 依托单位: MIDWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6754128
• 关键词: Escherichia coli; aminopterin; anaerobic bacteria; bacterial genetics; chemical kinetics; enzyme activity; folate; glutamates; methotrexate; microorganism growth; p aminobenzoate; protein purification; protein structure function; radiotracer; starvation; transport proteins; vitamin metabolism

DESCRIPTION (provided by applicant): Reduced derivatives of folic acid are involved in the biosynthesis of cellular components, including DNA, RNA, and protein. Essential in the human diet, folate is synthesized de novo in microorganisms. Conventional wisdom and early experiments supported the idea that many bacteria, including Escherichia coli (E. coli), are unable to import and utilize exogenous folate. This explained why the sulfonamide antibiotics, structural analogs of p-aminobenzoic acid (PABA), effectively inhibited folate biosynthesis and bacterial replication, despite residing in a human host containing folate. We have found that E. coli contains abgT, a cryptic gene for a transport protein that, when expressed in elevated levels, imparts two phenotypes: a highly increased sensitivity to the folate analogs, aminopterin and methotrexate, and the ability for a PABA auxotroph to grow on nanomolar quantities of the folate breakdown product, p-aminobenzoyl glutamate (PABA-GLU). PABA-GLU is a major catabolite of folate in humans, and is excreted in both urine and feces. The identification of AbgT as a possible transporter for either folate or folate catabolites (PABA-GLU) raises the possibility that E. coli possesses the ability to import folate and/or its breakdown products. This may occur in response to growth conditions that have not been identified previously. Folate transport and catabolism have remained largely unrecognized and unstudied in E. coli. This research proposal is designed to characterize this transport system. Specifically, we propose to characterize the transport protein AbgT with regard to its ability to take in aminopterin, methotrexate, folate, and PABA-GLU. We also intend to investigate a range of growth conditions to determine what might induce expression of abgT in wild-type cells; growth conditions to be investigated include nutrient starvation, anaerobic growth, and alterations in media pH. We also plan to purify and characterize the enzyme that cleaves PABA-GLU to form PABA, and to identify the gene associated with this activity. Finally, we plan to construct a strain of E. coli that can grow on physiologic concentrations of exogenous folate. This will likely entail optimizing transport of folate as well as reduction of folate to dihydrofolate. Successful completion of these studies will advance our understanding of basic metabolism and catabolism of the important vitamin folic acid in E. coli, especially with regard to a newly identified transport protein AbgT.

描述(申请人提供)：叶酸的还原衍生物参与细胞成分的生物合成，包括DNA、RNA和蛋白质。叶酸是人类饮食中必不可少的物质，在微生物中从头合成。传统智慧和早期实验支持这样一种观点，即包括大肠杆菌在内的许多细菌无法进口和利用外源叶酸。这就解释了为什么磺胺类抗生素，即对氨基苯甲酸(PABA)的结构类似物，尽管存在于含有叶酸的人体宿主中，仍能有效地抑制叶酸的生物合成和细菌复制。我们发现，大肠杆菌中含有AbgT，这是一种运输蛋白的隐藏基因，当表达水平升高时，会带来两种表型：对叶酸类似物氨基蝶呤和甲氨蝶呤的敏感性高度增加，以及PABA营养缺陷体能够依靠叶酸分解产物对氨基苯甲酰谷氨酸(PABA-GLU)的纳摩尔量生长。PABA-GLU是人类体内叶酸的一种主要分解代谢产物，在尿液和粪便中均有排泄。AbgT可能是叶酸或叶酸分解代谢产物的转运体(PABA-GLU)，这增加了大肠杆菌具有输入叶酸和/或其分解产物的能力的可能性。这可能是对以前没有确定的生长条件的反应。在大肠杆菌中，叶酸的运输和分解代谢在很大程度上仍未被认识和研究。这项研究方案旨在描述这种运输系统的特征。具体地说，我们建议根据转运蛋白AbgT摄取氨基蝶呤、甲氨蝶呤、叶酸和PABA-GLU的能力来表征该转运蛋白。我们还打算调查一系列的生长条件，以确定什么可能诱导AbgT在野生型细胞中表达；将被调查的生长条件包括营养饥饿、厌氧生长和培养基pH的变化。我们还计划纯化和鉴定裂解PABA-GLU形成PABA的酶，并鉴定与这一活性相关的基因。最后，我们计划构建一种能在生理浓度的外源叶酸上生长的大肠杆菌菌株。这可能需要优化叶酸的运输以及将叶酸还原为二氢叶酸。这些研究的成功完成将促进我们对重要的维生素叶酸在大肠杆菌中的基本代谢和分解代谢的理解，特别是关于新发现的转运蛋白AbgT。