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A radical new paradigm for heme degradation in enteric pathogens

肠道病原体血红素降解的全新范例

基本信息

批准号：
9906908
负责人：
WILLIAM N LANZILOTTA
金额：
$ 30.68万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-15 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9906908
关键词：
5&apos -deoxyadenosine Address Aerobic Anabolism Anaerobic Bacteria Animal Model Area Biliverdin reductase Binding Biochemical Biological Assay Biophysics Carbon Catalysis Cells Cessation of life Chemicals Coupled Crystallization Data Degradation Pathway Diet Disease Distal Environment Enzyme Kinetics Enzymes Escherichia coli Escherichia coli EHEC Escherichia coli O157:H7 Future Genes Goals Health Heart Heme Heme Iron Hemolytic-Uremic Syndrome Hemorrhagic colitis Homeostasis Human Hybrids Infection Intestines Investigation Iron Iron Chelation Kidney Failure Kinetics Metal Ion Binding Methyltransferase Modernization Modification Molecular NADP Organism Outcome Oxides Oxygen Pathogenicity Pathway interactions Play Porphyrins Process Production Property Proteins Protocols documentation Reaction Regulation Role S-Adenosylhomocysteine Source Structure Techniques Tetrapyrroles Vibrio cholerae Virulence Work antimicrobial antineoplastic antibiotics antitumor agent base cyclopropane drug development enteric pathogen experimental study in vitro Assay insight member novel pathogen pathogenic bacteria protein complex protoporphyrin IX public health relevance structural biology tool tumor

项目摘要

PROJECT SUMMARY For pathogens, the ability to acquire iron is critical and one of the best-understood indicators of virulence. Numerous pathogenic organisms take advantage of the abundance of heme in the host cell and the human diet as a source of essential iron. Until recently however, all of the known heme degrading enzymes required molecular oxygen for function. In this project, we will investigate the mechanism of anaerobic heme degradation in enterohemorrhagic E. coli (EHEC), a facultative anaerobic pathogen (e.g. O157:H7, which causes bloody diarrhea, hemolytic uremic syndrome, kidney failure and death). We recently found that the ChuW enzyme from EHEC catabolyzes heme and liberates iron under strictly anaerobic conditions. This newly identified anaerobic heme degradation enzyme is part of an important module of heme utilization proteins (ChuW, X, Y in E. coli) that is also found in other aggressive pathogens such as Vibrio cholerae. Identification and characterization of this new pathway in enteric pathogens provides an unexplored opportunity for developing novel antimicrobial compounds. In addition, the enzyme at the heart of this investigation (ChuW) is a radical SAM methyltransferase (RSMT); it utilizes a [4Fe-4S] cluster to generate a powerful radical species that facilitates the liberation of iron from heme through a methyl transfer reaction and chemical rearrangement of the porphyrin. ChuW is capable of methylating an otherwise unreactive sp2- hybridized carbon atom. Furthermore, ChuW is a member of the class C RSMTs, which are poorly understood but have already been shown to catalyze key reactions in the biosynthesis of novel compounds with antitumor and antibiotic properties. This work will provide new insight into this important class of RSMTs and how they control highly reactive radicals to facilitate specific chemical conversions in the biosynthesis of compounds including anti-microbial and anti-tumor agents. Using a multifaceted approach that combines traditional enzyme kinetics, rigorous spectroscopic techniques, and modern structural biology tools, we will characterize the mechanism of anaerobic heme degradation by ChuW, the properties of the anaerobic catabolites, and the interplay of two additional proteins with ChuW in the anaerobic degradation of heme as well as the transport and further reduction of the catabolites. The latter is important given recent work showing that heme degradation products play a vital role in regulation of heme flux and iron homeostasis in other aggressive pathogens. Our specific aims are to determine whether: ChuW catalyzes the liberation of iron from heme via a radical SAM methyltransferase mechanism (using 5'-dAdo• and resulting in formation of “anaerobilin”) (Aim 1); ChuX facilitates the chelation of the iron atom and delivery of “anaerobilin” to ChuY (Aim 3); and ChuY catalyzes the NADPH-dependent reduction of anaerobilin to “anaerorubin” (Aim 2). Our long term goal is to provide the necessary mechanistic insight to selectivity target the anaerobic heme degradation pathway, which appears to be specifically associated with a select set of aggressive, opportunistic, and pathogenic bacteria.

项目概要对于病原体来说，获取铁的能力至关重要，也是最容易理解的毒力指标之一。许多病原生物利用宿主细胞和人体中丰富的血红素饮食作为必需铁的来源。然而，直到最近，所有已知的血红素降解酶都需要分子氧的功能。在这个项目中，我们将研究厌氧血红素的机制肠出血性大肠杆菌 (EHEC) 的降解，这是一种兼性厌氧病原体（例如 O157:H7，导致血性腹泻、溶血性尿毒症综合征、肾衰竭和死亡）。我们最近发现来自肠出血性大肠杆菌的 ChuW 酶在严格厌氧条件下催化血红素并释放铁。这新发现的厌氧血红素降解酶是血红素利用的重要模块的一部分蛋白质（大肠杆菌中的 ChuW、X、Y）也存在于其他侵袭性病原体（例如霍乱弧菌）中。肠道病原体中这一新途径的鉴定和表征提供了一个未经探索的途径开发新型抗菌化合物的机会。此外，酶是这一切的核心研究 (ChuW) 是一种自由基 SAM 甲基转移酶 (RSMT)；它利用 [4Fe-4S] 簇来生成强大的自由基物质，通过甲基转移反应促进铁从血红素中释放，卟啉的化学重排。 ChuW 能够甲基化原本不反应的 sp2- 杂化碳原子。此外，ChuW 是 C 类 RSMT 的成员，但对其了解甚少但已被证明可以催化抗肿瘤新型化合物生物合成中的关键反应和抗生素特性。这项工作将为这一类重要的 RSMT 以及它们如何控制高活性自由基以促进化合物生物合成中的特定化学转化包括抗微生物剂和抗肿瘤剂。使用结合传统的多方面方法酶动力学、严格的光谱技术和现代结构生物学工具，我们将表征 ChuW 厌氧血红素降解机制、厌氧分解代谢物的特性以及两种附加蛋白质与 ChuW 在血红素厌氧降解以及运输中的相互作用并进一步减少分解代谢物。后者很重要，因为最近的研究表明血红素降解产物在其他侵袭性疾病中血红素通量和铁稳态的调节中发挥着至关重要的作用病原体。我们的具体目标是确定：ChuW 是否通过催化从血红素中释放铁激进的 SAM 甲基转移酶机制（使用 5'-dAdo• 并导致“厌氧蛋白”的形成）（目标 1）； ChuX 促进铁原子的螯合并将“厌氧素”传递给 ChuY（目标 3）；和楚Y 催化 NADPH 依赖性厌氧素还原为“厌氧红素”（目标 2）。我们的长期目标是为选择性靶向厌氧血红素降解途径提供必要的机制见解，该途径似乎与一组选定的侵袭性、机会性和致病性细菌有特定的相关性。