Biomaterial Database

Purification and characterization of nitrogenase from a delta nifW strain of Azotobacter vinelandii. 1994

S Kim, and B K Burgess

Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.

Deletion of the nifW gene in Azotobacter vinelandii yields a strain (DJ224) that grows poorly under N2 fixing conditions (Jacobson, M. R., Cash, V. L., Weiss, M. C., Laird, N. F., Newton, W. E., and Dean, D. R. (1989) Mol. & Gen. Genet. 219, 49-57). Here we report the purification of nitrogenase from DJ224. The purified Fe protein was indistinguishable from wild-type. The MoFe protein was indistinguishable from the wild-type MoFe protein by the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, native gel electrophoresis, two-dimensional gel electrophoresis, metal analysis, UV/visible and EPR spectroscopies. It was different by the criteria of CD spectroscopy and specific activities. At a 5:1 molar ratio of Fe protein to MoFe protein, H2 evolution under argon was identical to wild-type, C2H2 reduction was inhibited by 27%, N2 reduction was inhibited by 38%, and CO inhibited H2 evolution by 17%. The above data show that the nifW gene product is not required for: 1) detectable alteration of the polypeptide; 2) the synthesis of the metal portion of FeMo cofactor; or 3) FeMo cofactor insertion. The MoFe protein synthesized in the absence of NifW appears to have an alteration near the FeMo cofactor site, possibly at homocitrate, which causes differential inhibition of different substrates.

UI	MeSH Term	Description	Entries
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D008983	Molybdoferredoxin	A non-heme iron-sulfur protein isolated from Clostridium pasteurianum and other bacteria. It is a component of NITROGENASE, which is active in nitrogen fixation, and consists of two subunits with molecular weights of 59.5 kDa and 50.7 kDa, respectively.	Molybdenum-Iron Protein,FeMo Cofactor,Iron-Molybdenum Cofactor,MoFe Protein,Iron Molybdenum Cofactor,Molybdenum Iron Protein
D009586	Nitrogen Fixation	The process in certain BACTERIA; FUNGI; and CYANOBACTERIA converting free atmospheric NITROGEN to biologically usable forms of nitrogen, such as AMMONIA; NITRATES; and amino compounds.	Diazotrophy,Diazotrophic Activity,Dinitrogen Fixation,N2 Fixation,Activities, Diazotrophic,Activity, Diazotrophic,Diazotrophic Activities,Fixation, Dinitrogen,Fixation, N2,Fixation, Nitrogen
D009591	Nitrogenase	An enzyme system that catalyzes the fixing of nitrogen in soil bacteria and blue-green algae (CYANOBACTERIA). EC 1.18.6.1.	Dinitrogenase,Vanadium Nitrogenase,Nitrogenase, Vanadium
D002384	Catalysis	The facilitation of a chemical reaction by material (catalyst) that is not consumed by the reaction.	Catalyses
D002942	Circular Dichroism	A change from planar to elliptic polarization when an initially plane-polarized light wave traverses an optically active medium. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)	Circular Dichroism, Vibrational,Dichroism, Circular,Vibrational Circular Dichroism
D004578	Electron Spin Resonance Spectroscopy	A technique applicable to the wide variety of substances which exhibit paramagnetism because of the magnetic moments of unpaired electrons. The spectra are useful for detection and identification, for determination of electron structure, for study of interactions between molecules, and for measurement of nuclear spins and moments. (From McGraw-Hill Encyclopedia of Science and Technology, 7th edition) Electron nuclear double resonance (ENDOR) spectroscopy is a variant of the technique which can give enhanced resolution. Electron spin resonance analysis can now be used in vivo, including imaging applications such as MAGNETIC RESONANCE IMAGING.	ENDOR,Electron Nuclear Double Resonance,Electron Paramagnetic Resonance,Paramagnetic Resonance,Electron Spin Resonance,Paramagnetic Resonance, Electron,Resonance, Electron Paramagnetic,Resonance, Electron Spin,Resonance, Paramagnetic
D005798	Genes, Bacterial	The functional hereditary units of BACTERIA.	Bacterial Gene,Bacterial Genes,Gene, Bacterial
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001426	Bacterial Proteins	Proteins found in any species of bacterium.	Bacterial Gene Products,Bacterial Gene Proteins,Gene Products, Bacterial,Bacterial Gene Product,Bacterial Gene Protein,Bacterial Protein,Gene Product, Bacterial,Gene Protein, Bacterial,Gene Proteins, Bacterial,Protein, Bacterial,Proteins, Bacterial