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

• 批准号: 9929890
• 负责人: WILLIAM N LANZILOTTA
• 金额: $ 3.45万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9929890
• 关键词: 5' -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

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.

项目摘要 对于病原体来说，获得铁的能力至关重要，也是最好理解的毒力指标之一。 许多病原生物体利用宿主细胞和人类细胞中丰富的血红素， 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。在本计画中，我们将探讨厌氧血红素的作用机制 肠出血性E.大肠杆菌（EHEC），兼性厌氧病原体（例如O 157：H7， 导致出血性腹泻、溶血性尿毒综合征、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分解血红素并释放铁。这 新发现的厌氧血红素降解酶是血红素利用的重要模块之一 蛋白质（E.大肠杆菌），这也发现在其他侵略性病原体，如霍乱弧菌。 肠道病原体中这种新途径的鉴定和表征提供了一个未探索的 开发新型抗菌化合物的机会。此外，酶的核心， 研究（ChuW）是一种自由基SAM甲基转移酶（RSMT）;它利用[4Fe-4S]簇生成一个 通过甲基转移反应促进铁从血红素中释放的强大自由基物质， 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RSMTs的成员，这是知之甚少 但已经显示出催化具有抗肿瘤活性的新化合物的生物合成中的关键反应。 和抗生素特性。这项工作将提供新的见解，这一重要类别的RSMTs，以及他们如何 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。采用多方面的方法，结合传统的 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 本文综述了ChuW厌氧降解血红素的机理、厌氧催化剂的性质以及 在血红素的厌氧降解和运输中，两种额外的蛋白质与ChuW的相互作用 并进一步减少催化剂。后者很重要，因为最近的研究表明，血红素 降解产物在调节血红素通量和铁稳态中起着至关重要的作用， 病原体我们的具体目标是确定是否：ChuW催化铁从血红素中释放，通过 自由基SAM甲基转移酶机制（使用5 '-dAdo·并导致“厌氧菌素”的形成）（目的1）; ChuX促进铁原子的螯合和“厌氧菌素”向ChuY的递送（目的3）;和ChuY 催化厌氧菌素还原为“厌氧红蛋白”的NADPH依赖性还原（目的2）。我们的长期目标是 提供了必要的机制洞察力，以选择性靶向厌氧血红素降解途径， 似乎与一组选定的侵袭性、机会性和致病性细菌特别相关。