Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The catalytic mechanism for aerobic formation of methane by bacteria

Abstract

Methane is a potent greenhouse gas that is produced in significant quantities by aerobic marine organisms1. These bacteria apparently catalyse the formation of methane through the cleavage of the highly unreactive carbon–phosphorus bond in methyl phosphonate (MPn), but the biological or terrestrial source of this compound is unclear2. However, the ocean-dwelling bacterium Nitrosopumilus maritimus catalyses the biosynthesis of MPn from 2-hydroxyethyl phosphonate3 and the bacterial C–P lyase complex is known to convert MPn to methane4,5,6,7. In addition to MPn, the bacterial C–P lyase complex catalyses C–P bond cleavage of many alkyl phosphonates when the environmental concentration of phosphate is low4,5,6,7. PhnJ from the C–P lyase complex catalyses an unprecedented C–P bond cleavage reaction of ribose-1-phosphonate-5-phosphate to methane and ribose-1,2-cyclic-phosphate-5-phosphate. This reaction requires a redox-active [4Fe–4S]-cluster and S-adenosyl-l-methionine, which is reductively cleaved to l-methionine and 5′-deoxyadenosine8. Here we show that PhnJ is a novel radical S-adenosyl-l-methionine enzyme that catalyses C–P bond cleavage through the initial formation of a 5′-deoxyadenosyl radical and two protein-based radicals localized at Gly 32 and Cys 272. During this transformation, the pro-R hydrogen from Gly 32 is transferred to the 5′-deoxyadenosyl radical to form 5′-deoxyadenosine and the pro-S hydrogen is transferred to the radical intermediate that ultimately generates methane. A comprehensive reaction mechanism is proposed for cleavage of the C–P bond by the C–P lyase complex that uses a covalent thiophosphate intermediate for methane and phosphate formation.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: EPR spectra of wild-type PhnJ and the Cys272Ala mutant.
Figure 2: Mass spectra of 5′-deoxyadenosine and methane from reactions catalysed by PhnJ.
Figure 3: Identification of Gly 32 as the site of the glycyl radical.
Figure 4: Proposed mechanism for the reaction catalysed by PhnJ.
Figure 5: 31P-NMR spectrum of tryptic fragments of PhnJ after reaction with 2-dPRPn.

References

  1. Reeburgh, W. S. Ocean methane biogeochemistry. Chem. Rev. 107, 486–513 (2007)

    Article  CAS  PubMed  Google Scholar 

  2. Karl, D. M. et al. Aerobic production of methane in the sea. Nature Geosci. 1, 473–478 (2008)

    Article  ADS  CAS  Google Scholar 

  3. Metcalf, W. W. et al. Synthesis of methylphosphonic acid by marine microbes: a source of methane in the aerobic ocean. Science 337, 1104–1107 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wackett, L. P., Shames, S. L., Venditti, C. P. & Walsh, C. T. Bacterial carbon-phosphorus lyase: production, rates and regulation of phosphonic and phosphinic acid metabolism. J. Bacteriol. 169, 710–717 (1987)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Frost, J. W., Loo, S., Cordiero, M. & Li, D. Radical-based dephosphorylation and organophosphonate biodegradation. J. Am. Chem. Soc. 109, 2166–2171 (1987)

    Article  CAS  Google Scholar 

  6. Wackett, L. P., Wanner, B. L., Venditti, C. P. & Walsh, C. T. Involvement of the phosphate regulon and the psiD locus in the carbon-phosphorus lyase activity of Escherichia coli K-12. J. Bacteriol. 169, 1753–1756 (1987)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Metcalf, W. W. & Wanner, B. L. Mutational analysis of an Escherichia coli fourteen-gene operon for phosphonate degradation using TnphoA’ elements. J. Bacteriol. 175, 3430–3442 (1993)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kamat, S. S., Williams, H. J. & Raushel, F. M. Intermediates in the transformation of phosphonates to phosphate by bacteria. Nature 480, 570–573 (2011)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Cicchillo, R. M. et al. Escherichia coli lipoyl synthase binds two distinct [4Fe-4S] clusters per polypeptide. Biochemistry 43, 11770–11781 (2004)

    Article  CAS  PubMed  Google Scholar 

  10. Cicchillo, R. M. et al. Escherichia coli quinolinate synthetase does indeed harbor a [4Fe-4S] cluster. J. Am. Chem. Soc. 127, 7310–7311 (2005)

    Article  CAS  PubMed  Google Scholar 

  11. McGlynn, S. E. et al. Identification and characterization of a novel member of the radical AdoMet enzyme superfamily and implications for the biosynthesis of the Hmd hydrogenase active site cofactor. J. Bacteriol. 192, 595–598 (2010)

    Article  CAS  PubMed  Google Scholar 

  12. Zhang, Y. et al. Diphthamide biosynthesis requires an organic radical generated by an iron-sulphur enzyme. Nature 465, 891–896 (2010)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sofia, H. J., Chen, G., Hetzler, B. G., Reyes-Spindola, J. F. & Miller, N. E. Radical SAM, a novel superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visual methods. Nucleic Acids Res. 29, 1097–1106 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Frey, P. A., Hegeman, A. D. & Ruzicka, F. J. The radical SAM superfamily. Crit. Rev. Biochem. Mol. Biol. 43, 63–88 (2008)

    Article  CAS  PubMed  Google Scholar 

  15. Booker, S. J. & Grove, T. L. Mechanistic and functional versatility of radical SAM enzymes. F1000 Biol. Rep. 2, 52 (2010)

    Google Scholar 

  16. Eklund, H. & Fontecave, M. Glycyl radical enzymes: a conservative structural basis for radicals. Structure 7, R257–R262 (1999)

    Article  CAS  PubMed  Google Scholar 

  17. Logan, D. T., Andersson, J., Sjoberg, B. M. & Nordlund, P. A glycyl radical site in the crystal structure of a class III ribonucleotide reductase. Science 283, 1499–1504 (1999)

    Article  ADS  CAS  PubMed  Google Scholar 

  18. Becker, A. et al. Structure and mechanism of the glycyl radical enzyme pyruvate formate-lyase. Nature Struct. Biol. 6, 969–975 (1999)

    Article  CAS  PubMed  Google Scholar 

  19. Vey, J. L. et al. Structural basis for glycyl radical formation by pyruvate formate-lyase activating enzyme. Proc. Natl Acad. Sci. USA 105, 16137–16141 (2008)

    Article  ADS  CAS  PubMed  Google Scholar 

  20. Ghanem, E., Li, Y., Xu, C. & Raushel, F. M. Characterization of a phosphodiesterase capable of hydrolyzing EA 2192, the most toxic degradation product of the nerve agent VX. Biochemistry 46, 9032–9040 (2007)

    Article  CAS  PubMed  Google Scholar 

  21. Frey, M., Rothe, M., Wagner, A. F. V. & Knappe, J. Adenosyl methionine-dependent synthesis of the glycyl radical in pyruvate formate-lyase by abstraction of the glycine C-2 pro-S hydrogen atom. J. Biol. Chem. 269, 12432–12437 (1994)

    CAS  PubMed  Google Scholar 

  22. Licht, S., Garfen, G. J. & Stubbe, J. Thiyl radicals in ribonucleotide reductases. Science 271, 477–481 (1996)

    Article  ADS  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank D. Barondeau for use of the anaerobic chamber, R. Stipanovic for use of the gas chromatography mass spectrometer, A. Mehta and T. Begley for use of the liquid chromatography mass spectrometer, and C. Hilty for use of the 31P-NMR spectrometer. We thank P. A. Lindahl for help with the EPR measurements (GM084266). This work was supported by the Robert A. Welch Foundation (A-840).

Author information

Authors and Affiliations

Authors

Contributions

S.S.K., H.J.W. and F.M.R. designed the experiments. S.S.K. did the cloning and purification, performed the reactions and made all samples for analysis. S.S.K. and H.J.W. did the NMR, gas chromatography and gas chromatography mass spectrometry experiments. M.C. collected and analysed the EPR data. S.S.K. and L.J.D. did the trypsin digestion and peptide analysis. The manuscript was written by S.S.K. and F.M.R.

Corresponding author

Correspondence to Frank M. Raushel.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-8, Supplementary Text and Supplementary References. (PDF 801 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kamat, S., Williams, H., Dangott, L. et al. The catalytic mechanism for aerobic formation of methane by bacteria. Nature 497, 132–136 (2013). https://doi.org/10.1038/nature12061

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature12061

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing