Assembly and Maturation of the Iron-Sulfur Clusters of Nitrogenases

固氮酶铁硫簇的组装和成熟

• 批准号: 311061829
• 负责人: Professor Dr. Oliver Einsle
• 金额: --
• 依托单位: Institut für Biochemie
• 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 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/311061829?language=en
• 关键词: Assembly; Maturation; Iron; Sulfur; Clusters

The nitrogenase system is a two-component enzyme machinery that catalyzes the reductive fixation of atmospheric dinitrogen as bioavailable ammonium. Its subcomplexes, the ATP-hydrolyzing Fe protein and the catalytic dinitrogenase, contain complex iron-sulfur clusters that require a dedicated, multi-step maturation machinery for their assembly. While the sequence of steps in nitrogenase cofactor maturation is largely understood, current knowledge about the molecular details and mechanisms of the individual factors remains scarce. With the ability to generate variants and deletion strains of the model diazotroph Azotobacter vinelandii, we will address these open question in the present proposal. We focus on the three major steps of nitrogenase cofactor maturation, the NifSU complex that builds a basic [4Fe:4S] fragment for further assembly, the radical/SAM enzyme NifB that rearranges two such clusters into a complex cofactor precursor, and the EN scaffold that completes maturation by inserting an apical heterometal, Mo or V. Affinity-tagged constructs for the three complexes are available of will be generated during the project, to be investigated by X-ray crystallography and spectroscopy, making use of the broad range of methods and tools within PP 1927. In addition, we will use spatially refined anomalous dispersion (SpReAD) refinement to gain experimental insights into several of the complex metal clusters that are investigated within the consortium.

固氮酶系统是一种双组分酶机械，可催化大气中的二氮还原固定为生物可利用的铵。其子复合物，ATP 水解铁蛋白和催化二氮酶，含有复杂的铁硫簇，需要专用的多步骤成熟机器来进行组装。虽然固氮酶辅因子成熟的步骤顺序已基本被了解，但目前关于各个因子的分子细节和机制的知识仍然很少。凭借产生模型固氮菌固氮菌模型变体和缺失菌株的能力，我们将在本提案中解决这些悬而未决的问题。我们重点关注固氮酶辅因子成熟的三个主要步骤：构建基本 [4Fe:4S] 片段以供进一步组装的 NifSU 复合物、将两个此类簇重新排列成复杂辅因子前体的自由基/SAM 酶 NifB 以及 EN 支架通过插入顶端异金属、Mo 或 V 来完成成熟。项目期间将生成三种复合物的亲和标记结构，并通过 X 射线晶体学进行研究光谱学，利用 PP 1927 中的广泛方法和工具。此外，我们将使用空间精细异常色散 (SpReAD) 细化来获得对联盟内研究的几种复杂金属簇的实验见解。