National Academy of Sciences, Proceedings of the National Academy of Sciences, 37(117), p. 23165-23173, 2020
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Significance Engineering nitrogenase in plants may help alleviate economic and environmental issues due to the use of nitrogen fertilizer. Mitochondria have shown promise in supporting the function of nitrogenase, including electron donation and metallocluster assembly. Despite these successes, formation of the catalytic unit, NifDK, has proven difficult. Here, we find that when relocated to plant mitochondria, NifD is subject to errant peptidase-based cleavage and is insoluble. Guided by NifD sequence variation amongst bacteria and structural modeling, we designed NifD variants that avoided cleavage and retained function in bacterial assays. Fusion of NifK to degradation-resistant NifD also improved solubility, and the polyprotein retained function in bacterial assays. This work advances efforts to produce crops less reliant on nitrogen fertilizer.