Regulation of Bacterial Heme Biosynthesis

细菌血红素生物合成的调控

• 批准号: 6839927
• 负责人: MARK R O'BRIAN
• 金额: $ 25.23万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6839927
• 关键词: DNA binding protein; SDS polyacrylamide gel electrophoresis; bacterial proteins; biological signal transduction; enzyme induction /repression; ferrochelatase; gel mobility shift assay; genetic regulation; heme; laboratory rabbit; matrix assisted laser desorption ionization; oxidation; polymerase chain reaction; porphyrin biosynthesis; protein protein interaction; proteolysis

DESCRIPTION (provided by applicant): Heme is essential for many cellular processes, including oxidative stress responses, detoxification, respiration, signal transduction, and can also serve as a regulatory molecule to affect gene expression. Both the control of heme synthesis and its regulatory function must be reconciled with the fact that heme and its precursors are toxic as free molecules, and iron may be a limiting nutrient. The broad objective of the proposed work is to elucidate the control of bacterial heme biosynthesis, and to reconcile regulatory functions for heme and its precursors with their toxicity in ceils. The heme biosynthetic pathway culminates with the insertion of iron into protoporphyrin catalyzed by ferrochelatase. The Irr protein from the bacterium Bradyrhizobium japonicum represses the pathway at an early step under iron limitation to prevent protoporphyrin synthesis from exceeding iron availability. Data suggest that Irr interacts directly with ferrochelatase, and responds to iron via the status of heme and protoporphyrin localized at the site of synthesis. In the presence of iron, ferrochelatase inactivates Irr, followed by heme-dependent Irr degradation to derepress the pathway. Under iron limitation, protoporphyrin relieves the inhibition of Irr by ferrochelatase, probably by promoting protein dissociation, allowing genetic repression. The proposal addresses the hypothesis that metabolic control of the heme pathway involves a direct input signal from a biosynthetic enzyme to a regulator to affect gene expression. Furthermore, heme can serve as a signaling molecule without accumulating freely in cells, irr gene homologs are found in numerous pathogens and symbionts of eukaryotes, suggesting lrr-type regulation in other bacteria. Three specific aims are proposed to address the model. 1. Elucidate the mechanism by which heme promotes iron dependent degradation of Irr. Irr is a conditionally stable protein that involves direct binding of heme to the protein. We propose experiments to address the role of heme with an emphasis on exploring heme-mediated oxidation and proteolysis. 2. Characterize interactions between ferrochelatase and Irr. This interaction allows Irr to respond to the status of heme synthesis and is essential for control of lrr degradation and activity. Experiments are designed to further characterize the interaction and to elucidate the molecular basis of the inhibition of lrr activity by ferrochelatase. 3. Characterize Irr regulatory function with respect to its DNA-binding properties, functional domains and gene targets.

描述（由申请人提供）：血红素对许多细胞过程至关重要，包括氧化应激反应、解毒、呼吸、信号转导，也可以作为调节分子影响基因表达。血红素合成的控制及其调节功能必须与血红素及其前体作为游离分子是有毒的这一事实相协调，并且铁可能是限制性营养素。本研究的主要目的是阐明细菌血红素生物合成的调控机制，并将血红素及其前体的调控功能与它们在细胞中的毒性相协调。 血红素生物合成途径的高潮是铁螯合酶催化铁插入原卟啉。来自细菌慢生根瘤菌（Bradyrhizobium japonicum）的Irr蛋白在铁限制下在早期抑制该途径，以防止原卟啉合成超过铁可用性。数据表明，Irr直接与亚铁螯合酶相互作用，并通过血红素和原卟啉定位在合成位点的状态对铁作出反应。在铁的存在下，亚铁螯合酶失活Irr，随后通过血红素依赖性Irr降解去抑制途径。在铁限制下，原卟啉解除铁螯合酶对Irr的抑制，可能是通过促进蛋白质解离，允许遗传抑制。该提案解决了血红素途径的代谢控制涉及从生物合成酶到调节器的直接输入信号以影响基因表达的假设。此外，血红素可以作为一种信号分子，在细胞中不自由积累，irr基因同源物在许多病原体和真核生物的共生体中被发现，这表明在其他细菌中存在lrr类型的调节。 提出了三个具体目标，以解决该模型。1.阐明血红素促进Irr铁依赖性降解的机制。Irr是一种条件稳定的蛋白质，涉及血红素与蛋白质的直接结合。我们提出的实验，以解决血红素的作用，探索血红素介导的氧化和蛋白水解的重点。2.表征铁螯合酶和Irr之间的相互作用。这种相互作用使得Irr能够响应血红素合成的状态，并且对于控制lrr降解和活性是必不可少的。实验旨在进一步表征相互作用，并阐明铁螯合酶抑制lrr活性的分子基础。3.描述Irr在DNA结合特性、功能域和基因靶点方面的调控功能。