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制，催化还原固定大气中的二氮作为生物可利用的铵。它的亚复合物，ATP水解铁蛋白和催化二氮酶，含有复杂的铁硫簇，需要一个专门的，多步骤的成熟机制，他们的组装。虽然固氮酶辅因子成熟的步骤顺序在很大程度上是了解，目前的知识的分子细节和机制的个别因素仍然很少。随着产生模式固氮菌棕色固氮菌的变体和缺失菌株的能力，我们将在本提案中解决这些未决问题。我们专注于固氮酶辅因子成熟的三个主要步骤，NifSU复合物构建一个基本的[4Fe：4S]片段用于进一步组装，自由基/SAM酶NifB将两个这样的簇重新排列成复杂的辅因子前体，以及EN支架，通过插入顶端杂金属，Mo或V完成成熟。此外，我们将使用空间细化的异常色散（SpReAD）的细化，以获得实验的洞察到几个复杂的金属簇内的财团进行调查。