Crosstalk of iron-sulfur cluster assembly, metal homeostasis and the biosynthesis of molybdoenzymes

铁硫簇组装的串扰、金属稳态和钼酶的生物合成

• 批准号: 310702454
• 负责人: Professorin Dr. Silke Leimkühler
• 金额: --
• 依托单位: Arbeitsgruppe für Molekulare Enzymologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/310702454?language=en
• 关键词: Crosstalk; iron; sulfur; cluster; assembly

In recent years it has become evident that the biosynthesis of the molybdenum cofactor (Moco) and the assembly of iron-sulfur (Fe-S) clusters are directly connected to each other. In Escherichia coli, Moco biosynthesis thereby directly depends on the presence of Fe-S clusters or components of the Fe-S cluster assembly machinery at several levels. In the first step of Moco biosynthesis, the MoaA protein requires two [4Fe-4S] clusters for activity. Further, most molybdoenzymes in E. coli harbor numerous Fe-S clusters that are involved in intramolecular electron transfer reactions and are essential for the activity of the enzymes. In addition, the L-cysteine desulfurase IscS as major player for Fe-S cluster assembly has also been identified to mobilize the sulfur for the synthesis of the dithiolene group present in Moco. In addition, the expression of most molybdoenzymes and proteins involved in Moco biosynthesis is regulated by the transcriptional regulator for fumarate and nitrate reduction, FNR. The activity of FNR itself is directly dependent on the availability of Fe-S clusters under anaerobic conditions, consequently, Moco is not synthesized and molybdoenzymes are not expressed when Fe-S clusters are not assembled.The main questions to be solved are how iron availability and Fe-S cluster assembly regulates Moco biosynthesis and molybdoenzyme activity. The studies will be performed on the protein level in addition to the gene regulation level. We will unravel the complex regulatory network of molybdoenzymes by FNR. Specifically, we plan to decipher the intracellular iron trafficking and Fe-S cluster handover to FNR and selected molybdoenzymes. Particular aims are to identify the proteins that specifically insert [2Fe-2S] clusters and [4Fe-4S] clusters into molybdoenzymes. Novel factors that regulate gene expression on the level of translation efficiency of selected molybdoenzymes in dependence on the sulfur- and iron/Fe-S cluster availability will be identified. One particular focus is to study the regulation of the operon encoding TMAO reductase in E. coli, since we identified a novel Moco and iron-dependent expression of the torCAD genes.Studies on the level of gene regulation and complementing proteomic and metallomic studies including methods like high resolution clear native electrophoresis, 2D blue native nLC-MS/MS, whole cell Mössbauer spectroscopy and EPR spectroscopy, in addition to protein-protein interaction studies using microscale thermophoresis and fluorescence resonance energy transfer (FRET) will be applied within the collaborative framework of the SPP. In total, our analyses will shed light into the complex Fe-S cluster network that is essential for the production of active molybdoenzymes in E. coli.

近年来，已证明钼辅因子（Moco）的生物合成和铁硫（FE-S）簇的组装直接相互连接。在大肠杆菌中，Moco生物合成因此直接取决于Fe-S簇或Fe-S簇组装机械的成分的存在。在MOCO生物合成的第一步中，MOAA蛋白需要两个[4FE-4S]簇进行活性。此外，大肠杆菌中的大多数钼酶都有许多参与分子内电子转移反应的Fe-S簇，对于酶的活性至关重要。此外，还确定了L-半胱氨酸脱硫酶ISC作为Fe-S簇组装的主要参与者，以动员硫，以合成MOCO中存在的二硫代基团。此外，大多数钼酶和参与Moco生物合成的蛋白质的表达受到富马酸盐和硝酸盐还原的转录调节剂的调节，FNR。 FNR本身的活性直接取决于在厌氧条件下Fe-S簇的可用性，因此，当未组装Fe-S簇时，未合成Moco并未表达钼酶。要解决的主要问题。除基因调节水平外，还将对蛋白质水平进行研究。我们将通过FNR阐明钼酶的复杂调节网络。具体而言，我们计划破译细胞内铁运输和FE-S群集移交给FNR，并选择了钼酶。特定的目的是鉴定专门插入[2FE-2S]簇和[4FE-4S]簇中的蛋白质中。将确定基因表达在依赖于硫和铁/Fe-S簇的依赖性的转化效率水平上的基因表达的新因素。 One particular focus is to study the regulation of the operan encoding TMAO reduces in E. coli, since we identified a novel Moco and iron-dependent expression of the torCAD genes.Studies on the level of gene regulation and compiling proteomic and metallomic studies including methods like high resolution clear native electrophoresis, 2D blue native nLC-MS/MS, whole cell Mössbauer spectroscopy and EPR除了使用显微镜嗜热和荧光共振能量转移（FRET）的蛋白质 - 蛋白质相互作用研究外，光谱法还将在SPP的协作框架内应用。总的来说，我们的分析将使复杂的Fe-S群集网络浮出水面，该网络对于在大肠杆菌中生产活性钼酶至关重要。