Regulation of Bacterial Heme Biosynthesis

细菌血红素生物合成的调控

• 批准号: 6728795
• 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/6728795
• 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.

说明(申请人提供)：血红素对许多细胞过程是必不可少的，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来影响基因表达。对血红素合成的控制及其调节功能必须与以下事实相一致：作为自由分子，血红素及其前体是有毒的，而铁可能是一种限制性营养物质。这项拟议工作的广泛目标是阐明细菌血红素生物合成的控制，并协调血红素及其前体的调节功能与它们在细胞中的毒性。 血红素的生物合成途径在铁络合酶催化下将铁插入到原卟啉中达到顶峰。来自慢生根瘤菌的irr蛋白在铁限制的早期阶段抑制该途径，以防止原卟啉的合成超过铁的可利用性。数据表明，irr直接与铁络合酶相互作用，并通过位于合成部位的血红素和原卟啉的状态对铁作出反应。在铁存在的情况下，铁络合酶使irr失活，然后依赖于血红素的irr降解来解除该途径的抑制。在铁限制下，原卟啉可能通过促进蛋白质解离而解除铁络合酶对IRR的抑制，从而允许遗传抑制。该提议解决了这样的假设，即对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来影响基因表达。此外，血红素可以作为一种信号分子而不是在细胞内自由积累，irr基因同源物在许多真核生物的病原体和共生体中发现，这表明在其他细菌中存在LRR类型的调控。 提出了解决该模型的三个具体目标。1.阐明了血红素促进铁依赖的IRR降解的机制。IRR是一种条件稳定的蛋白质，涉及到血红素与蛋白质的直接结合。我们建议进行实验来解决血红素的作用，重点是探索血红素介导的氧化和蛋白质分解。2.研究铁络合酶与IRR之间的相互作用。这种相互作用使IRR能够对血红素合成状态做出反应，并且对于控制LRR的降解和活性是必不可少的。实验旨在进一步表征这种相互作用，并阐明铁络合酶抑制LRR活性的分子基础。3.从其与DNA的结合特性、功能结构域和基因靶点等方面对IRR的调控功能进行了研究。