State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences & School of Advanced Agricultural Sciences, Peking University, 100871 Beijing, China.
Department of Molecular Microbiology, John Innes Centre, NR4 7UH Norwich, United Kingdom
Proc Natl Acad Sci U S A. 2020 Jul 14;117(28):16537-16545. doi: 10.1073/pnas.2002307117. Epub 2020 Jun 29.
Engineering biological nitrogen fixation in eukaryotic cells by direct introduction of genes requires elegant synthetic biology approaches to ensure that components required for the biosynthesis of active nitrogenase are stable and expressed in the appropriate stoichiometry. Previously, the NifD subunits of nitrogenase MoFe protein from and were found to be unstable in yeast and plant mitochondria, respectively, presenting a bottleneck to the assembly of active MoFe protein in eukaryotic cells. In this study, we have delineated the region and subsequently a key residue, NifD-R98, from that confers susceptibility to protease-mediated degradation in mitochondria. The effect observed is pervasive, as R98 is conserved among all NifD proteins analyzed. NifD proteins from four representative diazotrophs, but not their R98 variants, were observed to be unstable in yeast mitochondria. Furthermore, by reconstituting mitochondrial-processing peptidases (MPPs) from yeast, , , and in , we demonstrated that MPPs are responsible for cleavage of NifD. These results indicate a pervasive effect on the stability of NifD proteins in mitochondria resulting from cleavage by MPPs. NifD-R98 variants that retained high levels of nitrogenase activity were obtained, with the potential to stably target active MoFe protein to mitochondria. This reconstitution approach could help preevaluate the stability of Nif proteins for plant expression and paves the way for engineering active nitrogenase in plant organelles.
通过直接引入基因在真核细胞中工程化生物固氮需要巧妙的合成生物学方法，以确保合成活性氮酶所需的组件在适当的化学计量下稳定表达。以前，来自 和 的氮酶 MoFe 蛋白的 NifD 亚基分别在酵母和植物线粒体中不稳定，这成为在真核细胞中组装活性 MoFe 蛋白的瓶颈。在这项研究中，我们已经确定了一个区域，随后确定了一个关键残基 NifD-R98，它使 中的 NifD 亚基易受线粒体中蛋白酶介导的降解。观察到的效果是普遍的，因为 R98 在所有分析的 NifD 蛋白中都保守。来自四个代表固氮生物的 NifD 蛋白，但不是它们的 R98 变体，在酵母线粒体中不稳定。此外，通过在 中重新组装来自酵母的线粒体加工肽酶 (MPP) 、 、 和 ，我们证明 MPPs 负责 NifD 的切割。这些结果表明，MPPs 的切割导致了 NifD 蛋白在线粒体中稳定性的普遍影响。获得了保留高氮酶活性的 NifD-R98 变体，有可能将活性 MoFe 蛋白稳定地靶向线粒体。这种重建方法可以帮助预先评估氮酶在植物表达中的稳定性，并为在植物细胞器中工程化活性氮酶铺平道路。
