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Volume 150, Issue 1

electron-transferring flavoprotein N electrochemically couples pyruvate dehydrogenase complex activity to N fixation Free

Abstract

thermolabile point mutants unable to fix N at 42 °C were isolated and mapped to three, unlinked loci; from complementation tests, several mutants were assigned to the locus. Of these, two independent mutants carried missense substitutions in the product electron-transferring flavoprotein N (ETF) -subunit. Both thermolabile missense variants Y238H and D229G mapped to the ETF interdomain linker. Unlinked thermostable suppressors of these two missense mutants were identified and mapped to the gene, encoding dihydrolipoamide dehydrogenase (LpDH), immediately distal to the genes, which collectively encode the pyruvate dehydrogenase (PDH) complex. These two suppressor alleles encoded LpDH NAD-binding domain missense mutants G187S and E210G. Crude cell extracts of these double mutants showed 60–70 % of the wild-type PDH activity; neither double mutant strain exhibited any growth phenotype at the restrictive or the permissive temperature. The genetic interaction between two combinations of and missense alleles implies a physical interaction of their respective products, LpDH and ETF. Presumably, this interaction electrochemically couples LpDH as the electron donor to ETF as the electron acceptor, allowing PDH complex activity (pyruvate oxidation) to drive soluble electron transport via ETF to N, which acts as the terminal electron acceptor. If so, then, the PDH complex activity sustains N fixation both as the driving force for oxidative phosphorylation and as the metabolic electron donor.

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Keyword(s): ETF, electron-transferring flavoprotein , LpDH, dihydrolipoamide dehydrogenase , Nifts, N2 fixation temperature-sensitive and PDH, pyruvate dehydrogenase
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2004-01-01
2024-04-24
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References

  1. Arigoni F., Kaminski P. A., Hennecke H., Elmerich C. 1991; Nucleotide sequence of the fixABC region of Azorhizobium caulinodans ORS571: similarity of the fixB product with eukaryotic flavoproteins, characterization of fixX, and identification of nifW. Mol Gen Genet 225:514–520
     [Google Scholar]
  2. Byron C. M., Stankovich M. T., Husain M., Davidson V. L. 1989; Unusual redox properties of electron-transfer flavoprotein from Methylophilus methylotrophus. Biochemistry 28:8582–8587 [CrossRef]
     [Google Scholar]
  3. Corbin D., Barran L., Ditta G. 1983; Organization and expression of Rhizobium meliloti nitrogen fixation genes. Proc Natl Acad Sci U S A 80:3005–3009 [CrossRef]
     [Google Scholar]
  4. Dallo S. F., Kannan T. R., Blaylock M. W., Baseman J. B. 2002; Elongation factor Tu and E1 beta subunit of pyruvate dehydrogenase complex act as fibronectin binding proteins in Mycoplasma pneumoniae. Mol Microbiol 46:1041–1051 [CrossRef]
     [Google Scholar]
  5. Donald R. G. K., Raymond C. K., Ludwig R. A. 1985; Vector insertion mutagenesis of Rhizobium sp. strain ORS571: direct cloning of mutagenised DNA sequences. J Bacteriol 162:317–323
     [Google Scholar]
  6. Donald R. G. K., Nees D., Raymond C. K., Loroch A. I., Ludwig R. A. 1986; Three genomic loci encode Rhizobium sp. ORS571 N2 fixation genes. J Bacteriol 165:72–81
     [Google Scholar]
  7. Dusha I., Kovalenko S., Banfalvi Z., Kondorosi A. 1987; Rhizobium meliloti insertion element ISRm2 and its use for identification of the fixX gene. J Bacteriol 169:1403–1409
     [Google Scholar]
  8. Earl C. D., Ronson C. W., Ausubel F. M. 1987; Genetic and structural analysis of the Rhizobium meliloti fixA,fixB, fixC, and fixX genes. . J Bacteriol 169:1127–1136
     [Google Scholar]
  9. Evans D., Jones R., Woodley P., Robson R. 1988; Further analysis of nitrogen fixation (nif)genes in Azotobacter chroococcum: identification and expression in Klebsiella pneumoniae of nifS, nifV, nifM and nifB genes and localization of nifE/N-,nifU-,nifA- and fixABC-like genes. J Gen Microbiol 134:931–942
     [Google Scholar]
  10. Finocchiaro G., Ito M., Ikeda Y., Tanaka K. 1988; Molecular cloning and nucleotide sequence of cDNAs encoding the alpha-subunit of human electron transfer flavoprotein. J Biol Chem 263:15773–15780
     [Google Scholar]
  11. Goodman S. I., Axtell K. M., Bindoff L. A., Beard S. E., Gill R. E., Frerman F. E. 1994; Molecular cloning and expression of a cDNA encoding human electron transfer flavoprotein-ubiquinone oxidoreductase. Eur J Biochem 219:277–286 [CrossRef]
     [Google Scholar]
  12. Herrick K. R., Salazar D., Goodman S. I., Finocchiaro G., Bedzyk L. A., Frerman F. E. 1994; Expression and characterization of human and chimeric human Paracoccus denitrificans electron transfer flavoprotein. J Biol Chem 269:32239–32245
     [Google Scholar]
  13. Hill S., Kavanagh E. P. 1980; Roles of nifF and nifJ gene products in electron transport to nitrogenase inKlebsiella pneumoniae. J Bacteriol 141:470–475
     [Google Scholar]
  14. Imperial J., Shah V. K., Ugalde R. A., Ludden P. W., Brill W. J. 1987; Iron-molybdenum cofactor synthesis in Azotobacter vinelandii Nif− mutants. J Bacteriol 169:1784–1786
     [Google Scholar]
  15. Jones M., Basran J., Sutcliffe M. J., Gunter-Grossmann J., Scrutton N. S. 2000; X-ray scattering studies of Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein. Evidence for multiple conformational states and an induced fit mechanism for assembly with trimethylamine dehydrogenase. J Biol Chem 275:21349–21354 [CrossRef]
     [Google Scholar]
  16. Kaminski P. A., Mandon K., Arigoni F., Desnoues N., Elmerich C. 1991; Regulation of nitrogen fixation in Azorhizobium caulinodans: identification of a fixK-like gene, a positive regulator of nifA. Mol Microbiol 5:1983–1991 [CrossRef]
     [Google Scholar]
  17. Koike M., Shah P. C., Reed L. J. 1960; α-Ketoacid dehydrogenation complexes.III. Purification and properties of dihydrolipoic dehydrogenase of Escherichia coli. J Biol Chem 235:1939–1943
     [Google Scholar]
  18. Kwon D. K., Beevers H. 1992; Growth of Sesbania rostrata (brem) with stem nodules under controlled conditions. Plant Cell Environ 15:939–945 [CrossRef]
     [Google Scholar]
  19. Ludwig R. A. 1987; Gene tandem-mediated selection of coliphage λ-receptive Agrobacterium, Pseudomonas, and Rhizobium strains. . Proc Natl Acad Sci U S A 84:3334–3338 [CrossRef]
     [Google Scholar]
  20. MacNeil T., MacNeil D., Roberts G. P., Brill W. J. 1978; Fine-structure mapping and complementation analysis of nif (nitrogen fixation) genes in Klebsiella pneumoniae. J Bacteriol 136:253–266
     [Google Scholar]
  21. Mattevi A., Schierbeek A. J., Hol W. G. 1991; Refined crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii at 2·2 Å resolution: a comparison with the structure of glutathione reductase. J Mol Biol 220:975–994 [CrossRef]
     [Google Scholar]
  22. Mattevi A., Obmolova G., Sokatch J. R., Betzel C., Hol W. G. 1992; The refined crystal structure of Pseudomonas putida lipoamide dehydrogenase complexed with NAD+ at 2·45 Å resolution. Proteins 13:336–351 [CrossRef]
     [Google Scholar]
  23. McKean M. C., Beckman J. D., Frerman F. E. 1983; Subunit structure of electron transfer flavoprotein. J Biol Chem 258:1866–1870
     [Google Scholar]
  24. O'Neill H., Mayhew S. G., Butler G. 1998; Cloning and analysis of the genes for a novel electron-transferring flavoprotein from Megasphaera elsdenii. J Biol Chem 273:21015–21024 [CrossRef]
     [Google Scholar]
  25. Pauling D. C., Lapointe J. P., Paris C. M., Ludwig R. A. 2001; Azorhizobium caulinodans pyruvate dehydrogenase activity is dispensable for aerobic but required for microaerobic growth. Microbiology 147:2233–2245
     [Google Scholar]
  26. Pühler A., Burkhardt H. J., Klipp W. 1979; Cloning of the entire region for nitrogen fixation from Klebsiella pneumoniae on a multicopy plasmid vehicle in Escherichia coli.. Mol Gen Genet 176:17–24 [CrossRef]
     [Google Scholar]
  27. Randall-Hazelbauer L., Schwartz M. 1973; Isolation of the bacteriophage lambda receptor from Escherichia coli. J Bacteriol 116:1436–1446
     [Google Scholar]
  28. Roberts G. P., MacNeil T., MacNeil D., Brill W. J. 1978; Regulation and characterization of protein products coded by the nif(nitrogen fixation) genes of Klebsiella pneumoniae.. J Bacteriol 136:267–279
     [Google Scholar]
  29. Roberts D. L., Salazar D., Fulmer J. P., Frerman F. E., Kim J.-J. P. 1999; Crystal structure of Paracoccus denitrificans electron transfer flavoprotein: structural and electrostatic analysis of a conserved flavin-binding domain. Biochemistry 38:1977–1989 [CrossRef]
     [Google Scholar]
  30. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  31. Schwartz M. 1975; Reversible interaction between coliphage lambda and its receptor protein. J Mol Biol 99:185–201 [CrossRef]
     [Google Scholar]
  32. Simon R., Priefer U., Pühler A. 1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Technology 1:784–791 [CrossRef]
     [Google Scholar]
  33. Smith R. F., Smith T. F. 1990; Automatic generation of primary sequence patterns from sets of related protein sequences. Proc Natl Acad Sci U S A 87:118–122 [CrossRef]
     [Google Scholar]
  34. Weidenhaupt M., Rossi P., Beck C., Fischer H.-M., Hennecke H. 1996; Bradyrhizobium japonicum possesses two discrete sets of electron transfer flavoprotein genes:fixA, fixB and etfS, etfL. Arch Microbiol 165:169–178
     [Google Scholar]
  35. Zhu J., Brill W. J. 1981; Temperature sensitivity of the regulation of nitrogenase synthesis by Klebsiella pneumoniae. J Bacteriol 145:1116–1118
     [Google Scholar]
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Azorhizobium caulinodans electron-transferring flavoprotein N electrochemically couples pyruvate dehydrogenase complex activity to N2 fixation
Microbiology 150, 117 (2004); https://doi.org/10.1099/mic.0.26603-0
/content/journal/micro/10.1099/mic.0.26603-0
/content/journal/micro/10.1099/mic.0.26603-0
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