Structural basis of nitrogenase assembly and protection from oxygen

固氮酶组装的结构基础和氧气保护

• 批准号: 2283950
• 金额: --
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2283950
• 关键词: Structural; basis; nitrogenase; assembly; protection

Nitrogenase catalyses the kinetically challenging reduction of dinitrogen (N2) to ammonia (NH3). Diazotrophic microorganisms expressing nitrogenase provide the majority of bioavailable nitrogen in the nitrogen cycle. Rising fixed nitrogen demands for global food production are met by supplementing from nitrogen fertilizer obtained through the Haber-Bosch process, which is both polluting and expensive. Biological nitrogen fixation (BNF) requires the coordinated expression of ~20 accessory genes and the assembly of several complex metal cofactors which are irreversibly damaged by oxygen. We therefore have yet to succeed in synthetically engineering or heterologously expressing BNF in plants to boost crop-yields. The binding site of dinitrogen as well as the electronic structure and reactivity of the catalytic metal cluster of molybdenum-dependent nitrogenase are not fully understood. We have yet to determine the roles of several nif (nitrogen-fixation) genes involved in assembling nitrogenase, as well as several accesory genes associated with the genomic region. In addition to the most-studied molybdenum-dependent nitrogenase there are several alternative nitrogenases with unsual properties which are less well studied. My PhD will contribute to a better understanding of some of the less well-studied proteins associated with nitrogen fixation, particularly those involved in oxygen protection. Azotobacter is a model diazotroph with unusually high oxygen tolerance and amenability to genetic modification. In Azotobacter, molybdenum-dependent nitrogenase is conformationally protected from oxygen through the reversible binding of a protective ferredoxin (FeSII), forming a ternary complex which is oxygen-tolerant but inactive. In the Murray lab, we recently showed by crystallographic methods that the current hypothesis of the mechanism of FeSII binding involving a large confromational change is likely incorrect. The structure of the protective ternary complex has not been determined. On my PhD I will investigate the conformational protection of nitrogenase from oxygen by the structural charactisation of the nitrogenase:FeSII complex by single-particle electron microscopy. To this end I aim to isolate the labile native complex using partial purification by sucrose density gradients, co-immunoprecipitation or alternatively by affinity-purification of the his-tagged complex. I further aim to support our previous structural studies of the mechanism of FeSII binding by crystallising and structurally characterising several FeSII homologs with high sequence identity to FeSII, including those identified in Confluentimicrobium, Marinobacterium and Rhodobacter. I am also interested in purifying and characterising an unidentified protein of similar size to FeSII that is stably associated with the nitrogenase in Xanthobacter autotrophicus. Based on the unusually high oxygen tolerance of the nitrogenase this protein may might represent a new type of conformational protection and would be a fourth nitrogenase structural gene, which I aim to characterise using the above mentioned methods. I furthermore aim to study some of the less-well understood proteins involved in biosynthesis of metal clusters of molybdenum-dependent nitrogenase such as NifZ, NifT and NifW by heterologous expression, purification and structural characterisation. There are also several proteins associated with the nif region (or anf and vnf for the alternative nitrogenases) with unknown functions. For example, the oxidase Anf3 was recently characterised in this lab, while the roles of Anf1 and Anf2 remain to be determined and will contribute to our understanding of the iron-only nitrogenase.

固氮酶催化二氮 (N2) 还原为氨 (NH3) 的动力学挑战。表达固氮酶的固氮微生物提供氮循环中的大部分生物可利用氮。全球粮食生产不断增长的固定氮需求是通过补充通过哈伯-博世工艺获得的氮肥来满足的，该工艺既污染又昂贵。 生物固氮 (BNF) 需要约 20 个辅助基因的协调表达以及几种复杂金属辅助因子的组装，这些金属辅助因子会被氧不可逆地损坏。因此，我们尚未成功地在植物中合成工程或异源表达 BNF 以提高作物产量。二氮的结合位点以及钼依赖性固氮酶催化金属簇的电子结构和反应性尚未完全了解。我们尚未确定参与固氮酶组装的几个 nif（固氮）基因以及与基因组区域相关的几个辅助基因的作用。除了研究最多的钼依赖性固氮酶之外，还有几种具有不寻常特性的替代固氮酶，但研究较少。我的博士学位将有助于更好地了解一些研究较少的与固氮相关的蛋白质，特别是那些与氧保护有关的蛋白质。 固氮菌是一种模型固氮菌，具有异常高的耐氧性和基因改造能力。在固氮杆菌中，钼依赖性固氮酶通过保护性铁氧还蛋白 (FeSII) 的可逆结合，在构象上受到保护，免受氧的影响，形成耐氧但无活性的三元复合物。在 Murray 实验室，我们最近通过晶体学方法表明，目前涉及大构象变化的 FeSII 结合机制的假设可能是不正确的。保护性三元配合物的结构尚未确定。 在我的博士学位期间，我将通过单粒子电子显微镜对固氮酶：FeSII 复合物的结构表征来研究固氮酶免受氧气影响的构象保护。为此，我的目标是通过蔗糖密度梯度、免疫共沉淀或通过组氨酸标记复合物的亲和纯化进行部分纯化来分离不稳定的天然复合物。我进一步的目标是通过结晶和结构表征与 FeSII 具有高度序列同一性的几种 FeSII 同源物（包括在汇合微生物、海洋杆菌和红细菌中鉴定的同源物）来支持我们之前对 FeSII 结合机制的结构研究。 我还对纯化和表征一种与 FeSII 大小相似的未鉴定蛋白质感兴趣，该蛋白质与自养黄杆菌中的固氮酶稳定相关。基于固氮酶异常高的氧耐受性，该蛋白质可能代表一种新型构象保护，并且将是第四种固氮酶结构基因，我的目的是使用上述方法对其进行表征。 此外，我的目标是通过异源表达、纯化和结构表征来研究一些不太了解的蛋白质，这些蛋白质参与钼依赖性固氮酶金属簇的生物合成，例如 NifZ、NifT 和 NifW。还有一些与 nif 区域（或替代固氮酶的 anf 和 vnf）相关的功能未知的蛋白质。例如，该实验室最近对氧化酶 Anf3 进行了表征，而 Anf1 和 Anf2 的作用仍有待确定，这将有助于我们对纯铁固氮酶的理解。