Functional expression of the FeMo-cofactor-specific biosynthetic genes nifEN as a NifE-N fusion protein synthesizing unit in Azotobacter vinelandii.

The nifEN encodes an E2N2 tetrameric metalloprotein complex that serves as scaffold for assembly of the FeMo cofactor of nitrogenase. In most diazotrophs, the NifE and NifN are translated as separate polypeptides and then assembled into tetrameric E2N2 complex. However, in Anabaena variabilis which has two nif clusters that encode two different NifEN complexes, the NifEN2 is encoded by a single nifE-N like gene, which has high homology to the NifE at amino-terminus and to the NifN at the carboxy-terminus. These observations implied that a metalloprotein like NifEN can accommodate large variations in their amino acid composition and also in the way they are synthesized (as two separate proteins or as a single protein) and yet remain functional. In Azotobacter vinelandii NifE and NifN are synthesized separately. To test whether NifEN could retain its functionality when encoded by a single gene, we generated a translational fusion of the nifE and nifN genes of A. vinelandii that could encode a large NifE-N fusion protein. When expressed in the nifEN-minus strain of A. vinelandii, the nifE-N gene fusion could complement the NifEN function. Western blot analysis by using polyclonal NifEN antibodies revealed that the complementing nifEN product is a large NifE-N fusion protein unit. The fact that the gene fusion of nifE-N specifies a functional NifE-N fusion protein reflects that these metalloproteins can accommodate a wide range of flexibility in their gene organization, structure, and assembly.