title: NifH and NifM proteins interact as demonstrated by the yeast two-hybrid system.
abstract: Biological nitrogen fixation is catalyzed by nitrogenase, a two-component enzyme consisting of the MoFe protein and the Fe protein. Two genes are involved in the formation of active Fe protein: nifH encodes the structural polypeptide, while nifM specifies a stabilizing and activation function by yet unknown mechanisms. Our studies were directed to clarify whether the NifM exerts its function through physical protein-protein interaction with NifH. To accomplish this, we used the yeast two-hybrid system. The simultaneous expression of the GAL4 binding domain-nifH fusion and GAL4 activation domain-nifM fusion resulted in the successful activation of GAL4-responsive HIS3, ADE2, and lacZ reporter genes in the two-hybrid system used. The system was also used to evidence the potential for in vivo NifH and NifM self-association. The results obtained suggest that NifH and NifM form homomers and also associate in between to form higher order complexes, which may be needed to exert the effect of NifM on Fe protein stability and activity.
authors.affiliation: Department of Molecular Genetics, Institute of Molecular Biology, Sofia, 1113, Bulgaria. npetrova@obzor.bio21.bas.bg
authors.forename: N
authors.initials: N
authors.lastname: Petrova
authors.forename: L
authors.initials: L
authors.lastname: Gigova
authors.forename: P
authors.initials: P
authors.lastname: Venkov
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D009586:Nitrogen Fixation; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D010088:Oxidoreductases; C029130:nitrogenase reductase; D001616:beta-Galactosidase