Functional Expression of a Fusion-dimeric MoFe Protein of Nitrogenase in Azotobacter vinelandii

Abstract

The MoFe protein component of the complex metalloenzyme nitrogenase is an ␣2␤2 tetramer encoded by the nifD and the nifK genes. In nitrogen fixing organisms, the ␣ and ␤ subunits are translated as separate polypeptides and then assembled into tetrameric MoFe protein complex that includes two types of metal centers, the P cluster and the FeMo cofactor. In Azotobacter vinelandii, the NifEN complex, the site for biosynthesis of the FeMo cofactor, is an ␣2␤2 tetramer that is structurally similar to the MoFe protein and encoded as two separate polypeptides by the nifE and the nifN genes. In Anabaena variabilis it was shown that a NifE-N fusion protein encoded by translationally fused nifE and nifN genes can support biological nitrogen fixation. The structural similarity between the MoFe protein and the NifEN complex prompted us to test whether the MoFe protein could also be functional when synthesized as a single protein encoded by nifD-K translational fusion. Here we report that the NifD-K fusion protein encoded by nifD-K translational fusion in A. vinelandii is a large protein (as determined by Western blot analysis) and is capable of supporting biological nitrogen fixation. These results imply that the MoFe protein is flexible in that it can accommodate major structural changes and remain functional.The nitrogenase enzyme, which is responsible for conversion of atmospheric nitrogen to ammonia, is found in all diazotrophs. It is actually composed of two separately purified, oxygen-labile, metalloproteins designated the Fe protein and the MoFe protein (1-7). The Fe protein is a homodimer of two identical subunits encoded by the nifH. Both subunits are bridged by one 4Fe-4S metal center and contain two nucleotide (MgATP or MgADP)-binding sites (5, 8 -10). The Fe protein serves as the obligate electron donor to MoFe protein during catalysis in a MgATP-and reductant-dependent process (11,12). The MoFe protein exists as an ␣2␤2 tetramer of about 240 kDa in size, encoded by nifD and nifK genes, respectively (13,14). Each ␣␤ heterodimer subunit binds two unique metal clusters, the FeMo cofactor and the P cluster. The FeMo cofactor that serves as the site of dinitrogen binding and reduction by the enzyme is located in the ␣ subunit, and the P cluster, which is positioned at the interface of ␣ and ␤ subunits of the heterodimer, is believed to mediate electron transfer from the Fe protein to the FeMo cofactor (15-17). During catalysis, the Fe protein forms a complex with the MoFe protein and transfers one electron to the MoFe protein with concomitant hydrolysis of two ATPs per Fe-MoFe protein interaction. It is accepted that the electron flow is from the Fe protein cluster to the MoFe protein P cluster and then to the FeMo cofactor, which is the substrate-binding and reduction site (13, 18 -20).Molecular evolutionary history of the nifDK, based on comparative analysis of the amino acid sequence of NifD, NifK, NifE, and NifN peptides, suggested that the genes encoding the NifDK and NifEN constitute a novel paralogous gene fam...

Polyprotein strategy for stoichiometric assembly of nitrogen fixation components for synthetic biology

“…2 A ). To further attempt optimization of NifHDK levels, we also incorporated fusion proteins with different linkers guided by previous studies and natural existing examples ( 24 – 27 ). Fused NifD∼K proteins showed broad tolerance to different lengths of GGGGS linkers, with a maximum activity of 91% when 5× GGGGS linkers were added ( SI Appendix , Fig.…”

Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix

“…Furthermore, the linker itself was designed to allow sufficient flexibility for the two subunits to form the correct α 2 β 2 heterotetrameric structure required for catalysis. An earlier report demonstrated that a NifD-K translational fusion without such a flexible linker can impart a limited degree of function (Suh et al, 2003; Lahiri et al, 2005). Therefore, we anticipate our version of NifD-linker-K will at least functionally substitute for individual NifD and NifK expression, possibly with greater efficacy than previously demonstrated.…”

The NifX Protein is Involved in the Final Stages of FeMo-cofactor Transport to the MoFe Protein

“…The His121 ligand of NafY that is known to be important for FeMoco binding (11) is shown to coincide with the His35 residue of NifX reaction showed lower levels of 99 Mo label incorporated into dinitrogenase in comparison to the NifX containing reaction [31] . Evidence obtained through our two-hybrid data clearly indicates the presence of protein-protein interaction between the NifDK protein encoding a single fused unit of dinitrogenase [45] and NifX and between the NifK component of the dinitrogenase and NifX. To further analyze if the NifX protein possessed specific or separate domains that helped in its interaction with 300 bp of nifX, encoding the N-terminal half of the NifX protein (N1X) and the region spanning 216-477 bp of nifX, encoding the Cterminal half of NifX fusion) and pMH5002 (α-RNAP:NifX) was 45.24 ± 8.72 Miller Units and that of pBG1718 (λCI: NifK) and pMH5002 (α-RNAP:NifX) was 46.34 ± 8.81.…”