Department of Biochemistry, Virginia Techgrid.438526.e, Blacksburg, Virginia, USA.
Institute of Biochemistry, Albert-Ludwigs Universität, Freiburg, Germany.
mBio. 2021 Aug 31;12(4):e0156821. doi: 10.1128/mBio.01568-21. Epub 2021 Jul 20.
The nitrogen-fixing microbe Azotobacter vinelandii has the ability to produce three genetically distinct, but mechanistically similar, components that catalyze nitrogen fixation. For two of these components, the Mo-dependent and V-dependent components, their corresponding metal-containing active site cofactors, designated FeMo-cofactor and FeV-cofactor, respectively, are preformed on separate molecular scaffolds designated NifEN and VnfEN, respectively. From prior studies, and the present work, it is now established that neither of these scaffolds can replace the other with respect to their cofactor assembly functions. Namely, a strain inactivated for NifEN cannot produce active Mo-dependent nitrogenase nor can a strain inactivated for VnfEN produce an active V-dependent nitrogenase. It is therefore proposed that metal specificities for FeMo-cofactor and FeV-cofactor formation are supplied by their respective assembly scaffolds. In the case of the third, Fe-only component, its associated active site cofactor, designated FeFe-cofactor, requires neither the NifEN nor VnfEN assembly scaffold for its formation. Furthermore, there are no other genes present in A. vinelandii that encode proteins having primary structure similarity to either NifEN or VnfEN. It is therefore concluded that FeFe-cofactor assembly is completed within its cognate catalytic protein partner without the aid of an intermediate assembly site. Biological nitrogen fixation is a complex process involving the nitrogenases. The biosynthesis of an active nitrogenase involves a large number of genes and the coordinated function of their products. Understanding the details of the assembly and activation of the different nitrogen fixation components, in particular the simplest one known so far, the Fe-only nitrogenase, would contribute to the goal of transferring the necessary genetic elements of bacterial nitrogen fixation to cereal crops to endow them with the capacity for self-fertilization. In this work, we show that there is no need for a scaffold complex for the assembly of the FeFe-cofactor, which provides the active site for Fe-only nitrogenase. These results are in agreement with previously reported genetic reconstruction experiments using a non-nitrogen-fixing microbe. In aggregate, these findings provide a high degree of confidence that the Fe-only system represents the simplest and, therefore, most attractive target for mobilizing nitrogen fixation into plants.
固氮微生物 Azotobacter vinelandii 能够产生三种在遗传上不同但在机制上相似的催化固氮的成分。对于其中两种成分，即 Mo 依赖性和 V 依赖性成分，它们相应的含金属活性位点辅因子分别指定为 FeMo-辅因子和 FeV-辅因子，分别在分别指定的 NifEN 和 VnfEN 分子支架上预先形成。从前人的研究和本工作中可以确定，这两种支架都不能在其辅因子组装功能方面相互替代。即，失活 NifEN 的菌株不能产生活性的 Mo 依赖性氮酶，而失活 VnfEN 的菌株也不能产生活性的 V 依赖性氮酶。因此，提议 FeMo-辅因子和 FeV-辅因子形成的金属特异性由它们各自的组装支架提供。对于第三种，Fe 仅成分，其相关的活性位点辅因子，指定为 FeFe-辅因子，其形成既不需要 NifEN 也不需要 VnfEN 组装支架。此外，在 A. vinelandii 中没有其他基因编码与 NifEN 或 VnfEN 具有一级结构相似性的蛋白质。因此，可以得出结论，FeFe-辅因子的组装是在其同源催化蛋白伴侣内完成的，而不需要中间组装位点的帮助。
生物固氮是一个复杂的过程，涉及氮酶。活性氮酶的生物合成涉及大量基因及其产物的协调功能。了解不同固氮成分的组装和激活的细节，特别是迄今为止最简单的 Fe 仅氮酶，将有助于将细菌固氮所需的遗传元件转移到谷类作物中，赋予它们自花授粉的能力。在这项工作中，我们表明，FeFe-辅因子的组装不需要支架复合物，而 FeFe-辅因子为 Fe 仅氮酶提供了活性位点。这些结果与先前使用非固氮微生物进行的遗传重建实验结果一致。总的来说，这些发现提供了高度的信心，即 Fe 仅系统代表了最简单的、因此也是最有吸引力的将固氮转移到植物中的目标。
