Novel components of iron sulfur cluster biosynthesis pathways

描述（由申请人提供）：铁硫（FeS）簇是古代普遍存在的金属蛋白辅助因子，其起源被认为是早期厌氧生物圈的还原环境。向有氧生活的过渡给FeS团簇带来了几个问题，因为氧化限制了铁的生物利用度，并直接促进了团簇被氧物种（如超氧化物和过氧化物）破坏，这是有氧呼吸的副产物。宿主防御机制利用了这种易感性，将FeS集群作为促进细胞死亡的组成部分。FeS簇的成分铁和硫对细胞具有高毒性，并且FeS簇是通过特定的生物合成途径形成的，例如大肠杆菌中的iscRSUhscBAfdx操纵子和sufABCDSE操纵子。我们的长期目标是确定一套完整的参与原核模型大肠杆菌中FeS簇生物合成的因素，并从功能上表征它们在FeS簇形成中的作用。本研究的目的是表征一种新的因子，cgfs型单硫醇戊二醇还蛋白（GrxD），它与大肠杆菌Suf - FeS生物合成途径有关，并确定其在大肠杆菌中的作用。Suf通路似乎已经适应了应激条件下FeS簇合成的作用，如氧化应激和铁限制，并且已被证明是植物病原体Erwinia菊花的完全毒力所必需的。此外，FeS簇生物合成过程是一个重要的特征，Suf系统已被确定为某些细菌物种中唯一的FeS簇生物合成途径，例如结核病的病原体结核分枝杆菌。结核病每年导致大约200万人死亡，主要发生在发展中国家。近年来，Suf系统已经有了一些详细的特征，然而，对Isc和Suf系统的研究尚未确定使Suf能够在应激条件下合成FeS簇的具体适应性。此外，最近发现的这两个核心操纵子之外参与FeS簇生物合成的因子表明，其他未编码在Suf操纵子中的因子，如GrxD，可能与Suf系统协同工作，促进胁迫条件下FeS簇的合成。利用生化分析、功能基因组学、蛋白质化学和分子生物学的结合，我们的目标是(i)确定grxD和Isc系统突变体之间合成致死率的基础，以及这些遗传相互作用是否可以在特定条件下减轻；（ii）表征grxD的物理相互作用，确定其参与的蛋白质复合物的组成，以及结合伙伴对grxD生化特性的影响。（iii）具体评估GrxD转移其FeS簇的能力，以及（iv）确定GrxD与Suf和Isc系统的功能关联。

DESCRIPTION (provided by applicant): Iron-sulfur (FeS) clusters are ancient ubiquitous metalloprotein cofactors whose origins are thought to lay in the reducing environment of the early anaerobic biosphere. The transition to aerobic life has created several problems for FeS clusters by both limiting the bio-availability of iron due to oxidation and directly promoting the destruction of clusters with oxygen species such as superoxide and peroxide, which are by-products of aerobic respiration. This susceptibility has been leveraged by host defense mechanisms, which target FeS clusters as components through which to promote cell death. The components of FeS clusters, iron and sulfur, are highly toxic to cells and FeS clusters are formed by specific biosynthesis pathways such as the iscRSUhscBAfdx operon and sufABCDSE operon in Escherichia coli. Our long term goal is to determine a complete set of factors involved in FeS cluster biosynthesis in the model prokaryote E. coli and to functionally characterize their roles in FeS cluster formation. The objective of the proposed research is the characterization of a novel factor, the CGFS-type monothiol glutaredoxin, GrxD, which has been implicated as functioning with the E. coli Suf FeS biosynthesis pathway, and the determination of its role in E. coli. The Suf pathway appears to have adapted to a role in FeS cluster synthesis under stress conditions, such as oxidative stress and iron limitation, and has been shown to be necessary for complete virulence of the plant pathogen Erwinia chrysanthemi. Moreover, the process of FeS cluster biosynthesis is an essential trait, and the Suf system has been identified as the only FeS cluster biosynthesis pathway in some bacterial species, such as the pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis. Tuberculosis kills approximately 2 million people a year, mainly in the developing world. The Suf system has been characterized in some detail in recent years, however, studies of both Isc and Suf systems have yet to identify specific adaptations which make Suf capable of FeS cluster synthesis under stress conditions. Moreover, recent identification of factors outside of these two core operons, which participate in FeS cluster biosynthesis, suggests that other factors not encoded in the Suf operon, such as GrxD, may work with the Suf system and facilitate the synthesis of FeS clusters under stress conditions. Using a combination of biochemical assays, functional genomics, protein chemistry and molecular biology our aims are to (i) determine the basis of synthetic lethality between grxD and Isc system mutants, and if these genetic interactions can be alleviated under specific conditions, (ii) characterize the physical interactions of GrxD and determine the composition of protein complexes in which it participates, and the effect of binding partners on the biochemical properties of GrxD, (iii) specifically assess the ability of GrxD to transfer its FeS cluster, and (iv) determine the functional association of GrxD to both Suf and Isc systems. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Iron sulfur clusters are essential for life, but are prone to oxidative damage and are targeted by mammalian systems to promote bacterial death. The Suf FeS cluster biosynthesis pathway has been adapted for synthesis under conditions of stress and may play a role in bacterial survival during virulence and is a potential target for novel antimicrobials. Our proposal is designed to characterize a novel factor which may work with the Suf system and may be responsible for its capacity for FeS synthesis under stress conditions.