Molecular and Cellular Biochemistry

, Volume 6, Issue 1, pp 53–64

Flavin mononucleotide reductase of luminous bacteria

  • Warren Duane
  • J. W. Hastings
Review and General Articles b. general articles

Summary

NAD(P)H: FMN oxidoreductase (flavin reductase) couplesin vitro to bacterial luciferase. This reductase, which is also postulated to supply reduced flavin mononucleotidein vivo as a substrate for the bioluminescent reaction, has been partially purified and characterized from two species of luminous bacteria. FromPhotobacterium fischeri the enzyme has a M.W. determined by Sephadex gel filtration, of 43,000 and may have a subunit structure. The turnover number at 20 °C, based on a purity estimate of 20%, is 1.7 × 104 moles of NADH oxidized per min per mole of reductase. The reductase isolated fromBeneckea harveyi has an apparent molecular weight of 23,000; its purity was too low to permit estimation of specific activity. Using a spectrophotometric assay at 340 nm with theP. fischeri reductase, both NADH (Km, 8 × 10−5m) and NADPH (Km, 4 × 10−4m) were enzymatically oxidized, the Vmax with NADH being approximately twice that of NADPH. Of the flavins tested in this assay, only FMN (Km, 7.3 × 10−5m) and FAD (Km, 1.4 × 10−4m) were effective, FMN having a Vmax three times that of FAD. In the coupled assay, i.e., measuring the bioluminescence intensity of the reaction with added luciferase, the optimum FMN concentration was nearly 100 times less than in the spectrophotometric assay. The studies reported suggest the existence of a functional reductaseluciferase complex.

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References

  1. 1.
    W. D. McElroy and A. Green, Arch. Biochem. Biophys. 56, 240–255 (1955).Google Scholar
  2. 2.
    K. H. Nealson and J. W. Hastings, J. Biol. Chem. 247, 888–894 (1972).Google Scholar
  3. 3.
    A. Eberhand and J. W. Hastings, Biochem. Biophys. Res. Comm. 47, 348–353 (1972).Google Scholar
  4. 4.
    O. Shimomura, F. H. Johnson and Y. Kohama, Proc. Nat. Acad. Sci. U.S.A. 69, 2086–2089 (1972).Google Scholar
  5. 5.
    F. McCapra and D. W. Hysert, Biochem. Biophys. Res. Comm. 52, 298–304 (1973).Google Scholar
  6. 6.
    D. K. Dunn, G. A. Michaliszyn, I. G. Bogacki, and E. A. Meighen, Biochemistry 12, 4911–4918 (1973).Google Scholar
  7. 7.
    B. L. Strehler and M. J. Cormier, Arch. Biochem. Biophys. 47, 16–33 (1953).Google Scholar
  8. 8.
    J. W. Hastings, W. H. Riley and J. Massa, J. Biol. Chem. 240, 1473–1481 (1965).Google Scholar
  9. 9.
    Q. H. Gibson and J. W. Hastings, Biochem. J. 83, 368–377 (1962).Google Scholar
  10. 10.
    M. J. Cormier and J. T. Totter, Biochem et Biophys. Acta 25, 229–237 (1957).Google Scholar
  11. 11.
    M. J. Cormier and S. Kuwabara, Photochem. Photobiol. 4, 1217–1225 (1965).Google Scholar
  12. 12.
    S. Kuwabara, M. J. Cormier, L. S. Dure, P. Kreiss, and P. Pfuderer, Proc. Nat. Acad. Sci. U.S.A. 53, 822–828 (1965).Google Scholar
  13. 13.
    Q. H. Gibson, J. W. Hastings, G. Weber, W. Duane, and J. Massa, in Flavins and Flavoproteins (Slater, E. C., editor) pp. 341–359, Elsevier Publishing Co., Amsterdam (1966).Google Scholar
  14. 14.
    A. Gunsalus-Miguel, E. A. Meighen, M. Nicoli, K. H. Nealson, and J. W. Hastings, J. Biol. Chem. 247, 398–404 (1972).Google Scholar
  15. 15.
    O. H. Lowry, N. A. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265–276 (1951).Google Scholar
  16. 16.
    O. Warburg and W. Christian, Biochem. Z. 310, 384–421 (1941).Google Scholar
  17. 17.
    J. W. Hastings and G. Weber, J. Opt. Soc. Am. 53, 1410–1415 (1963).Google Scholar
  18. 18.
    J. W. Hastings, K. Weber, J. Friedland, A. Eberland, G. W. Mitchell, and A. Gunsalus, Biochemistry 8, 4681–4689 (1969).Google Scholar
  19. 19.
    J. L. Reichelt and P. Baumann, Arch. Mikrobiol. 94, 283–330 (1973).Google Scholar
  20. 20.
    J. M. Friedland and J. W. Hastings, Proc. Nat. Acad. Sci. U.S.A. 58, 2336–2342 (1967).Google Scholar
  21. 21.
    D. Keilin and E. F. Hartree Proc. Roy. Soc. (London) Ser. B. 124, 397–405 (1938).Google Scholar
  22. 22.
    J. M. Friedland and J. W. Hastings, Biochemistry 6, 2893–2900 (1967).Google Scholar
  23. 23.
    W. C. Duane, PhD Thesis (University of Illinois, Urbana) (1969).Google Scholar
  24. 24.
    P. W. Trudgill, R. Dubus and I. C. Gunsalus, J. Biol. Chem. 241, 1194–1205 (1966).Google Scholar
  25. 25.
    Puget, K. and A. M. Michelson, Biochemie 54, 1197–1204 (1972).Google Scholar
  26. 26.
    W. D. McElroy, J. W. Hastings, V. Sonnenfeld, and J. Coulombre, J. Bacteriol. 67, 402–408 (1954).Google Scholar
  27. 27.
    J. J. Coffey, J. Bacteriol. 94, 1638–1647 (1967).Google Scholar
  28. 28.
    K. H. Nealson, T. Platt and J. W. Hastings, J. Bacteriol. 104, 313–322 (1970).Google Scholar
  29. 29.
    A. Eberhard, J. Bacteriol. 109, 1101–1105 (1972).Google Scholar
  30. 30.
    H. R. Mahler and E. H. Cordes, Biological Chemistry, Harper and Row Inc., New York (1966).Google Scholar

Copyright information

© Dr. W. Junk b.v. Publishers 1975

Authors and Affiliations

  • Warren Duane
    • 1
  • J. W. Hastings
    • 1
  1. 1.The Biological LaboratoriesHarvard UniversityCambridgeUSA
  2. 2.Diamond Shamrock Research Lab.PainesvilleUSA

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