Heme Biosynthesis in Bradyrhizobium japonicum

本项目的长期目标是了解共生固氮菌慢生根瘤菌中血红素的生物合成规律。含铁的卟啉环(血红素)参与了几个酶反应，需要微厌氧条件才能有效地固氮(自由或共生)。形成血红素的多酶途径以铁在铁络合酶催化的反应中插入原卟啉环而结束。由于铁可能是一种限制性营养物质，而卟啉具有细胞毒性，这表明需要协调血红素的生物合成与铁的有效性。最近，一种名为irr的新蛋白质被发现并被提出用于介导铁对血红素生物合成的控制。IRR是在铁限制下存在的，它负向调节血红素生物合成基因hemB，防止原卟啉积累超过铁的有效性。IRR是一种条件稳定的蛋白质，当细胞暴露在铁中时，它会迅速降解。在这种铁介导的降解过程中，血红素是一个效应器分子，与IRR中的特定基序结合。IRR还可直接与铁络合酶结合。这项提议将检验这样一种假设，即irr通过响应原卟啉和血红素在血红素合成部位的状态来介导铁对血红素途径的控制。血红素在调节铁依赖的irr降解中的作用将被阐明。具体地说，将测试两种看似合理的机制：血红素催化irr的氧化导致降解，或血红素诱导irr的构象变化，使其可被蛋白酶访问。还将研究铁络合酶与IRR的结合以及这种结合对IRR活性的影响。这项工作的重点是一种代谢途径，这对能量产生和细菌细胞对其新的调节非常重要，因此应该为细菌适应不同环境条件的能力提供见解。慢生大豆根瘤菌的环境适应性具有特殊的意义，因为这种细菌与大豆建立了固氮共生关系。由于这种关系对一种重要的商业作物有利，因此日本大豆具有经济和农业利益。这些研究还将涉及对研究生和本科生的培训，包括妇女和代表不足的少数族裔受训人员。

The long-term aim of this project is to understand the regulation of heme biosynthesis in the symbiotic nitrogen-fixing bacterium Bradyrhizobium japonicum. The iron-containing porphyrin ring (heme) is involved in several enzymatic reactions requiring microanaerobic conditions necessary for efficient nitrogen fixation (free-living or symbiotic). The multienzyme pathway for heme formation ends with the insertion of iron into the protoporphyrin ring in a reaction catalyzed by ferrochelatase. Because iron may be a limiting nutrient and porphyrins are cytotoxic, this suggests the need for coordination of heme biosynthesis with iron availability. A novel protein called Irr has been recently identified and proposed to mediate iron control of heme biosynthesis. Irr is present under iron limitation and negatively regulates the heme biosynthesis gene hemB, preventing protoporphyrin accumulation from exceeding iron availability. Irr is a conditionally stable protein that degrades rapidly when cells are exposed to iron. Heme is an effector molecule in this iron-mediated degradation, binding to a specific motif in Irr. Irr also binds ferrochelatase directly. This proposal will test the hypothesis that Irr mediates iron control of the heme pathway by responding to the status of protoporphyrin and heme at the site of heme synthesis. The role of heme in mediating iron-dependent Irr degradation will be elucidated. Specifically, two plausible mechanisms will be tested: heme catalysis of Irr oxidation leading to degradation, or heme-induced conformational change in Irr, rendering it accessible to proteases. The binding of ferrochelatase to Irr and the effect of this binding on Irr activity will also be studied. This work focuses on a metabolic pathway that is important for energy production and its novel regulation by bacterial cells, and thus should provide insights on the ability of bacteria to adjust to diverse environmental conditions. The environmental adaptability of Bradyrhizobium japonicum is of special interest because this bacterium establishes a nitrogen-fixing symbiotic association with soybeans. Because this relationship is beneficial to a commercially important plant crop, B. japonicum is thus of economic and agricultural interest. These studies will also involve the training of graduate and undergraduate students, including women and underrepresented minority trainees.