To engineer Mo-dependent nitrogenase function in plants, expression of the structural proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here, we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in Escherichia coli identified a subset that retained function, including a single amino acid variant (Y100Q). We found that other naturally occurring NifD proteins containing alternate amino acids at the Y100 position were also less susceptible to degradation. The Y100Q variant also enabled expression of a NifD(Y100Q)–linker–NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)–linker–NifK retained function in bacterial nitrogenase assays, demonstrating that this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.

为了工程化植物中依赖Mo的固氮酶功能，结构蛋白NifD和NifK的表达将是绝对必要的。尽管线粒体已被确立为氧敏感酶（如NifH）生物合成的合适真核环境，但由于隐蔽的NifD降解，已证明在该细胞器中NifD的表达十分困难。在这里，我们描述了此问题的解决方案。使用分子和蛋白质组学方法，我们发现NifD降解是NifD内特定基序的线粒体内切蛋白酶活性的结果。着眼于此功能敏感区域，我们设计了NifD变体，包括一个至三个氨基酸取代，并区分了几个在植物和酵母线粒体中均表达抗降解的变体。大肠杆菌确定了保留功能的子集，包括单个氨基酸变体（Y100Q）。我们发现在Y100位置含有其他氨基酸的其他天然NifD蛋白也较不易降解。Y100Q变体还能够在植物线粒体中表达NifD（Y100Q）-接头-NifK翻译多蛋白，这一点已通过在植物提取物可溶级分中鉴定出多蛋白而得以证实。NifD（Y100Q）–接头–NifK在细菌固氮酶测定中保留了功能，表明该多蛋白允许在定义的化学计量学中支持活性的NifD和NifK表达。我们的结果证明了蛋白质设计如何克服在新型环境中表达合成蛋白质时遇到的障碍。特别，