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. 1987 Oct;84(20):7066–7069. doi: 10.1073/pnas.84.20.7066

Site-directed mutagenesis of the nitrogenase MoFe protein of Azotobacter vinelandii

Kevin E Brigle *, Robert A Setterquist *, Dennis R Dean *, John S Cantwell , Mary C Weiss , William E Newton †,‡,§
PMCID: PMC299230  PMID: 16593879

Abstract

A strategy has been formulated for the site-directed mutagenesis of the Azotobacter vinelandii nifDK genes. These genes encode the α and β subunits of the MoFe protein of nitrogenase, respectively. Six mutant strains, which produce MoFe proteins altered in their α subunit by known single amino acid substitutions, have been produced. Three of these transversion mutations involve cysteine-to-serine changes (at residues 154, 183, and 275), two involve glutamine-to-glutamic acid changes (at residues 151 and 191), and one involves an aspartic acid-to-glutamic acid change (at residue 161). All three possible phenotypic responses are observed within this group— i.e., normal, slow, and no growth in the absence of a fixed-nitrogen source. Two-dimensional gel electrophoresis indicates that all mutants accumulate normal levels of the subunits of both nitrogenase component proteins. Whole-cell and crude-extract acetylene-reduction activities indicate substantial levels of Fe protein activity in all strains. In contrast, MoFe protein activities do not parallel the diazotrophic growth capability for all strains. Two strains appear to exhibit altered substrate discrimination. Such analyses should aid in the identification of metallocluster-binding sites and subunit-subunit interaction domains of the MoFe protein and also provide insight into the mechanistic roles of the various prosthetic groups in catalysis.

Keywords: iron-molybdenum cofactor, nitrogen fixation, nifD gene, protein engineering

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Selected References

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  1. Beynon J., Cannon M., Buchanan-Wollaston V., Cannon F. The nif promoters of Klebsiella pneumoniae have a characteristic primary structure. Cell. 1983 Sep;34(2):665–671. doi: 10.1016/0092-8674(83)90399-9. [DOI] [PubMed] [Google Scholar]
  2. Bishop P. E., Jarlenski D. M., Hetherington D. R. Evidence for an alternative nitrogen fixation system in Azotobacter vinelandii. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7342–7346. doi: 10.1073/pnas.77.12.7342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brigle K. E., Newton W. E., Dean D. R. Complete nucleotide sequence of the Azotobacter vinelandii nitrogenase structural gene cluster. Gene. 1985;37(1-3):37–44. doi: 10.1016/0378-1119(85)90255-0. [DOI] [PubMed] [Google Scholar]
  4. Brigle K. E., Weiss M. C., Newton W. E., Dean D. R. Products of the iron-molybdenum cofactor-specific biosynthetic genes, nifE and nifN, are structurally homologous to the products of the nitrogenase molybdenum-iron protein genes, nifD and nifK. J Bacteriol. 1987 Apr;169(4):1547–1553. doi: 10.1128/jb.169.4.1547-1553.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buchanan-Wollaston V., Cannon M. C., Cannon F. C. The use of cloned nif (nitrogen fixation) DNA to investigate transcriptional regulation of nif expression in Klebsiella pneumoniae. Mol Gen Genet. 1981;184(1):102–106. doi: 10.1007/BF00271203. [DOI] [PubMed] [Google Scholar]
  6. Burgess B. K., Stiefel E. I., Newton W. E. Oxidation-reduction properties and complexation reactions of the iron-molybdenum cofactor of nitrogenase. J Biol Chem. 1980 Jan 25;255(2):353–356. [PubMed] [Google Scholar]
  7. Dunham W. R., Hagen W. R., Braaksma A., Grande H. J., Haaker H. The importance of quantitative Mössbauer spectroscopy of MoFe-protein from Azotobacter vinelandii. Eur J Biochem. 1985 Feb 1;146(3):497–501. doi: 10.1111/j.1432-1033.1985.tb08679.x. [DOI] [PubMed] [Google Scholar]
  8. Hageman R. V., Burris R. H. Nitrogenase and nitrogenase reductase associate and dissociate with each catalytic cycle. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2699–2702. doi: 10.1073/pnas.75.6.2699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hawkes T. R., McLean P. A., Smith B. E. Nitrogenase from nifV mutants of Klebsiella pneumoniae contains an altered form of the iron-molybdenum cofactor. Biochem J. 1984 Jan 1;217(1):317–321. doi: 10.1042/bj2170317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Itakura K., Rossi J. J., Wallace R. B. Synthesis and use of synthetic oligonucleotides. Annu Rev Biochem. 1984;53:323–356. doi: 10.1146/annurev.bi.53.070184.001543. [DOI] [PubMed] [Google Scholar]
  11. Kennedy C., Robson R. L. Activation of nif gene expression in Azotobacter by the nifA gene product of Klebsiella pneumoniae. Nature. 1983 Feb 17;301(5901):626–628. doi: 10.1038/301626a0. [DOI] [PubMed] [Google Scholar]
  12. Kurtz D. M., Jr, McMillan R. S., Burgess B. K., Mortenson L. E., Holm R. H. Identification of iron-sulfur centers in the iron-molybdenum proteins of nitrogenase. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4986–4989. doi: 10.1073/pnas.76.10.4986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  14. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  15. Orme-Johnson W. H., Hamilton W. D., Jones T. L., Tso M. Y., Burris R. H., Shah V. K., Brill W. J. Electron paramagnetic resonance of nitrogenase and nitrogenase components from Clostridium pasteurianum W5 and Azotobacter vinelandii OP. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3142–3145. doi: 10.1073/pnas.69.11.3142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Roberts G. P., MacNeil T., MacNeil D., Brill W. J. Regulation and characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae. J Bacteriol. 1978 Oct;136(1):267–279. doi: 10.1128/jb.136.1.267-279.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Robinson A. C., Burgess B. K., Dean D. R. Activity, reconstitution, and accumulation of nitrogenase components in Azotobacter vinelandii mutant strains containing defined deletions within the nitrogenase structural gene cluster. J Bacteriol. 1986 Apr;166(1):180–186. doi: 10.1128/jb.166.1.180-186.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Shah V. K., Brill W. J. Isolation of an iron-molybdenum cofactor from nitrogenase. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3249–3253. doi: 10.1073/pnas.74.8.3249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Smith B. E., Lang G. Mössbauer spectroscopy of the nitrogenase proteins from Klebsiella pneumoniae. Structural assignments and mechanistic conclusions. Biochem J. 1974 Feb;137(2):169–180. doi: 10.1042/bj1370169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Smith B. E., Lowe D. J., Bray R. C. Studies by electron paramagnetic resonance on the catalytic mechanism of nitrogenase of Klebsiella pneumoniae. Biochem J. 1973 Oct;135(2):331–341. doi: 10.1042/bj1350331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Strandberg G. W., Wilson P. W. Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii. Can J Microbiol. 1968 Jan;14(1):25–31. doi: 10.1139/m68-005. [DOI] [PubMed] [Google Scholar]
  23. Watt G. D., Burns A., Tennent D. L. Stoichiometry and spectral properties of the MoFe cofactor and noncofactor redox centers in the MoFe protein of nitrogenase from Azotobacter vinelandii. Biochemistry. 1981 Dec 8;20(25):7272–7277. doi: 10.1021/bi00528a034. [DOI] [PubMed] [Google Scholar]
  24. Zimmermann R., Münck E., Brill W. J., Shah V. K., Henzl M. T., Rawlings J., Orme-Johnson W. H. Nitrogenase X: Mössbauer and EPR studies on reversibly oxidized MoFe protein from Azotobacter vinelandii OP. Nature of the iron centers. Biochim Biophys Acta. 1978 Dec 20;537(2):185–207. doi: 10.1016/0005-2795(78)90504-4. [DOI] [PubMed] [Google Scholar]
  25. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]

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