Mutagenesis of nifE and nifN from Azotobacter vinelandii
Wilson, Mark Steven Michael
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The products of nifE and nifN from Azotobacter vinelandii, which are involved in the biosynthesis of the iron-molybdenum cofactor (FeMo-co) co) from nitrogenase, have been analyzed using a variety of mutagenic techniques. NifE was the object of several site-specific, amino acid substitutions that were designed to elicit information regarding metal cluster ligands, subunit-subunit interactions, and the proposed transfer of FeMo-co.from a nifEN-products complex to the apo-MoFe protein. A model of metal cluster binding; regions within the nifEN-products is discussed insofar as it relates to the rationale for the targeting of particular amino acids for-substitution. A translational fusion between nifN and lacZ was constructed and used to study the regulation of nifEN. This gene fusion was regulated in the same manner as wild type nifN and produced a fusion protein which was enzymatically active with respect to substrates of Î²-galactosidase. Results from mutant strains which carry lesions in nifH or nifA in addition to the nifN::lacZ fusion are presented and discussed.
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