Engineering a functional NifDK polyprotein resistant to mitochondrial degradation

Abstract

To engineer Mo dependent nitrogenase function in plants expression of 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 E. coli identified a subset that retained function, including a single amino acid (Y100Q) variant. 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 E. coli based nitrogenase assays, demonstrating 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.

Using synthetic biology to overcome barriers to stable expression of nitrogenase in eukaryotic organelles

“…Although we considered that a number of candidate endopeptidases could be responsible for the degradation of NifD in the mitochondrial matrix, once we had identified the region required for cleavage, we focused on the possibility that the mitochondrial processing peptidase was carrying out secondary processing of NifD close to residue R98. Although there is significant degeneracy in the amino acid sequences recognized by MPPs, the importance of R98 and the positioning of an aromatic residue two residues downstream (Y100) was suggestive of cleavage by the MPP (38). The complete conservation of the residue equivalent to R98, in combination with the highly conserved Y100 residue in NifD proteins from diverse sources, implies that the degradation problem cannot be resolved by exploiting the biodiversity of NifD proteins (38).…”

“…Although there is significant degeneracy in the amino acid sequences recognized by MPPs, the importance of R98 and the positioning of an aromatic residue two residues downstream (Y100) was suggestive of cleavage by the MPP (38). The complete conservation of the residue equivalent to R98, in combination with the highly conserved Y100 residue in NifD proteins from diverse sources, implies that the degradation problem cannot be resolved by exploiting the biodiversity of NifD proteins (38). Indeed, we found this to be so when examining the stability of NifD from four different diazotrophs in yeast mitochondria and discovered that in each case, cleavage could be prevented by constructing the equivalent R98K substitution (Fig.…”

Transfer of Nitrogen Fixation (nif) Genes to Non‐diazotrophic Hosts

“…By design and mutation, NifD variants resistantt od egradation were obtained if expressed in both plant andy east mitochondria. [69] These studies established the feasibility of reconstituting the complete componentry of biosynthetic and catalytic nitrogenase in plant cells.…”