Plant expression of NifD protein variants resistant to mitochondrial degradation
Robert S. Allen, Christina M. Gregg, Shoko Okada, Amratha Menon, Dawar Hussain, Vanessa Gillespie, Ema Johnston, Rosangela Devilla, Andrew C. Warden, Matthew C. Taylor, Keren Byrne, Michelle L. Colgrave, Craig C. Wood
Abstract
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.