1887

Microbiology Society logo

Volume 138, Issue 7

Functional expression in of proteins B and C from soluble methane monooxygenase of (Bath) Free

Abstract

(Bath) uses a soluble methane monooxygenase (sMMO) to catalyse the oxidation of methane to methanol. sMMO is comprised of three components; A, B and C. Protein C (the reductase) transfers electrons from NADH to protein A (the hydroxylase) which contains the active site, and protein B regulates this electron flow. The five genes encoding the sMMO proteins and their subunits are clustered and have been cloned in . A DNA fragment containing , the gene encoding protein B, was subcloned into pT7–5, a plasmid of the T7 RNA polymerase promoter expression system. Upon induction, expressed protein B which was fully functional after purification. The gene encoding protein C, , was amplified with unique restriction sites at each end using the polymerase chain reaction and then subcloned into pT7–7 (a plasmid similar to pT7–5 but containing its own ribosome-binding site and ATG start codon). Protein C expressed in was also found to be functional. This is the first report of the functional expression of methanotroph methane monooxygenase genes in a heterologous host and represents a significant step forward in our analysis of the assembly and catalysis of sMMO.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
© Society for General Microbiology, 1992
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-138-7-1301
1992-07-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/138/7/mic-138-7-1301.html?itemId=/content/journal/micro/10.1099/00221287-138-7-1301&mimeType=html&fmt=ahah

References

  1. Anthony C. 1986; Bacterial oxidation of methane and methanol. Advances in Microbial Physiology 27 113 210
    [Google Scholar]
  2. Cardy D. L. N., Laidler V., Salmond G. P. C., Murrell J. C. 1991; Molecular analysis of the methane monooxygenase (MMO) gene cluster of Methylosinus trichosporium OB3b. Molecular Micro-biology 5 335 342
    [Google Scholar]
  3. Colby J., Dalton H. 1978; Resolution of the methane monooxygenase of Methylococcus capsulatus (Bath) into three components. Biochemical Journal 171 461 468
    [Google Scholar]
  4. Colby J., Stirling D., Dalton H. 1977; The soluble methane monooxygenase of Methylococcus capsulatus (Bath). Its ability to oxygenate n-alkanes, n-alkenes, ethers, alicyclic aromatic and heterocyclic compounds. Biochemical Journal 165 395 403
    [Google Scholar]
  5. Dalton H., Prior S. D., Leak D. J., Stanley S. 1984; Regulation and control of methane monooxygenase. In Microbial growth on C1 compounds pp. 75 82 Edited by Crawford R. L., Hanson R. S. Proceedings of the 4th International Symposium; Washington DC: American Society for Microbiology;
    [Google Scholar]
  6. Ericson A., Hedman B., Hodgson K. O., Green J., Dalton H., Bentsen J. G., Beer R. H., Lippard S. J. 1988; Structural characterization by EXAFS spectroscopy of the binuclear iron centre in protein A of methane monooxygenase from Methylococcus capsulatus (Bath). Journal of the American Chemical Society 110 2330 2332
    [Google Scholar]
  7. Goeddel D. V. 1990; Gene expression technology. Methods in Enzymology 185 60 89
    [Google Scholar]
  8. Green J., Dalton H. 1985; Protein B of the soluble methane monooxygenase of Methylococcus capsulatus (Bath): a novel regulatory protein of enzyme activity. Journal of Biological Chemistry 260 15795 15801
    [Google Scholar]
  9. Green J., Dalton H. 1986; Steady-state kinetic analysis of soluble methane monooxygenase from Methylococcus capsulatus (Bath). Biochemical Journal 236 155 162
    [Google Scholar]
  10. Harlow E., Lane D. 1988; Antibodies. A Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press;
    [Google Scholar]
  11. Hase T., Wakabayashi S., Matsubara H., Kerscher L., Oester-helt D., Rao K. K., Hall D. O. 1978; Complete amino acid sequence of Halobacterium halobium ferredoxin containing an NE acetyllysine residue. Journal of Biochemistry 83 1657 1670
    [Google Scholar]
  12. Hase T., Matsubara H., Hutber G. N., Rogers L. J. 1982; Amino acid sequences of Nostoc strain MAC ferredoxins I and II. Journal of Biochemistry 92 1347 1355
    [Google Scholar]
  13. Higgins I. J., Hammond R. C., Sariaslani F. S., Best D., Davies M. M., Tryhorn S. E., Taylor F. 1979; Biotransformation of hydrocarbons and related compounds by whole organism suspensions of methane-grown Methylosinus trichosporium OB3b. Biochemical and Biophysical Research Communications 89 671 677
    [Google Scholar]
  14. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227 680 685
    [Google Scholar]
  15. Lidstrom M. E., Stirling D. I. 1990; Methylotrophs: genetics and commercial applications. Annual Reviews of Microbiology 44 27 58
    [Google Scholar]
  16. Liu K. E., Lippard S. J. 1991; Redox properties of the hydroxylase component of methane monooxygenase from Methylococcus capsulatus (Bath). Journal of Biological Chemistry 266 12836 12839
    [Google Scholar]
  17. Lund J., Dalton H. 1985; Further characterization of the FAD and Fe2S2 redox centres of component C, the NADH: acceptor reductase of the soluble methane monooxygenase of Methylococcus capsulatus (Bath). European Journal of Biochemistry 147 291 296
    [Google Scholar]
  18. Lund J., Woodland M. P., Dalton H. 1985; Electron transfer reactions in the soluble methane monooxygenase of Methylococcus capsulatus (Bath). European Journal of Biochemistry 147 297 305
    [Google Scholar]
  19. Maniatis T., Fritsch E. F., Sambrook J. 1982 Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press;
    [Google Scholar]
  20. Mullens I. A., Dalton H. 1987; Cloning of the gamma-subunit methane monooxygenase from Methylococcus capsulatus. Biotechnology 5 490 493
    [Google Scholar]
  21. Pilkington S. J., Salmond G. P. C., Murrell J. C., Dalton H. 1990; Identification of the gene encoding the regulatory protein B of soluble methane monooxygenase. FEMS Microbiology Letters 72 345 348
    [Google Scholar]
  22. Prior S. D., Dalton H. 1985; The effect of copper ions on membrane content and methane monooxygenase activity in methanol-grown cells of Methylococcus capsulatus (Bath). Journal of General Microbiology 131 155 163
    [Google Scholar]
  23. Sjöberg B. M., Sanders-Loehr J., Loehr T. M. 1987; Identification of a hydroxide ligand at the iron centre of ribonucleotide reductase by resonance raman spectroscopy. Biochemistry 26 4242 4247
    [Google Scholar]
  24. Stainthorpe A. C., Murrell J. C., Salmond G. P. C., Dalton H., Lees V. 1989; Molecular analysis of methane monooxygenase from Methylococcus capsulatus (Bath). Archives of Microbiology 152 154 159
    [Google Scholar]
  25. Stainthorpe A. C., Lees V., Salmond G. P. C., Dalton H., Murrell J. C. 1990; The methane monooxygenase gene cluster of Methylococcus capsulatus (Bath). Gene 91 27 34
    [Google Scholar]
  26. Stanley S. H., Prior S. D., Leak D. J., Dalton H. 1983; Copper stress underlies the fundamental change in intracellular location of methane monooxygenase in methane-oxidizing organisms: studies in batch and continuous cultures. Biotechnology letters 5 487 492
    [Google Scholar]
  27. Tabor S. 1990; Expression using the T7 RNA polymerase/promoter system. In Current Protocols in Molecular Biology pp. 16.2.1 16.2.11 Edited by Ausubel F. A., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. New York: Greene Publishing and Wiley-Interscience;
    [Google Scholar]
  28. Takahashi Y., Hase T., Wada K., Matsubara H. 1983; Ferrodoxins in developing spinach cotyledons; the presence of two molecular species. Plant Cell Physiology 24 189 198
    [Google Scholar]
  29. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 76 4350 4354
    [Google Scholar]
  30. Woodland M. P., Dalton H. 1984; Purification and properties of component A of the methane monooxygenase from Methylococcus capsulatus (Bath). Journal of Biological Chemistry 259 53 59
    [Google Scholar]
  31. Yoch D. C., Carithers R. P. 1979; Bacterial iron–sulphur proteins. Microbiology Reviews 43 384 421
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-138-7-1301
Loading
Functional expression in Escherichia coli of proteins B and C from soluble methane monooxygenase of Methylococcus capsulatus (Bath)
Microbiology 138, 1301 (1992); https://doi.org/10.1099/00221287-138-7-1301
/content/journal/micro/10.1099/00221287-138-7-1301
/content/journal/micro/10.1099/00221287-138-7-1301
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error