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.

  • Article
  • Published:

The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases

Nature Chemical Biology volume 12, pages 298–303 (2016)Cite this article

Subjects

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery, LPMOs have become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here a structural determination of an LPMO-oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: LPMO activity and active site.
Figure 2: Ls(AA9)A and Ta(AA9)A activity on insoluble and soluble cellulose.
Figure 3: Structural views of Ls(AA9)A.
Figure 4: Structure of Ls(AA9)A around active site before and after binding of G3 (R = H).
Figure 5: EPR spectra of Ls(AA9)A at 150 K.

Similar content being viewed by others

Accession codes

Primary accessions

NCBI Reference Sequence

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Himmel, M.E. et al. Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315, 804–807 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Sheldon, R.A. Green and sustainable manufacture of chemicals from biomass: state of the art. Green Chem. 16, 950–963 (2014).

    Article  CAS  Google Scholar 

  3. Payne, C.M. et al. Fungal cellulases. Chem. Rev. 115, 1308–1448 (2015).

    Article  CAS  PubMed  Google Scholar 

  4. Beeson, W.T., Vu, V.V., Span, E.A., Phillips, C.M. & Marletta, M.A. Cellulose degradation by polysaccharide monooxygenases. Annu. Rev. Biochem. 84, 923–946 (2015).

    Article  CAS  PubMed  Google Scholar 

  5. Horn, S.J., Vaaje-Kolstad, G., Westereng, B. & Eijsink, V.G.H. Novel enzymes for the degradation of cellulose. Biotechnol. Biofuels 5, 45 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Müller, G., Várnai, A., Johansen, K.S., Eijsink, V.G.H. & Horn, S.J. Harnessing the potential of LPMO-containing cellulase cocktails poses new demands on processing conditions. Biotechnol. Biofuels 8, 187 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Hemsworth, G.R., Henrissat, B., Davies, G.J. & Walton, P.H. Discovery and characterization of a new family of lytic polysaccharide monooxygenases. Nat. Chem. Biol. 10, 122–126 (2014).

    Article  CAS  PubMed  Google Scholar 

  8. Lo Leggio, L. et al. Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase. Nat. Commun. 6, 5961 (2015).

    Article  CAS  PubMed  Google Scholar 

  9. Cantarel, B.L. et al. The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res. 37, D233–D238 (2009).

    Article  CAS  PubMed  Google Scholar 

  10. Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P.M. & Henrissat, B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42, D490–D495 (2014).

    Article  CAS  PubMed  Google Scholar 

  11. Merino, S.T. & Cherry, J. Progress and challenges in enzyme development for biomass utilization. Adv. Biochem. Eng. Biotechnol. 108, 95–120 (2007).

    CAS  PubMed  Google Scholar 

  12. Harris, P.V. et al. Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family. Biochemistry 49, 3305–3316 (2010).

    Article  CAS  PubMed  Google Scholar 

  13. Vaaje-Kolstad, G. et al. An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides. Science 330, 219–222 (2010).

    Article  CAS  PubMed  Google Scholar 

  14. Forsberg, Z. et al. Cleavage of cellulose by a CBM33 protein. Protein Sci. 20, 1479–1483 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Quinlan, R.J. et al. Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components. Proc. Natl. Acad. Sci. USA 108, 15079–15084 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Phillips, C.M., Beeson, W.T., Cate, J.H. & Marletta, M.A. Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa. ACS Chem. Biol. 6, 1399–1406 (2011).

    Article  CAS  PubMed  Google Scholar 

  17. Hemsworth, G.R., Davies, G.J. & Walton, P.H. Recent insights into copper-containing lytic polysaccharide mono-oxygenases. Curr. Opin. Struct. Biol. 23, 660–668 (2013).

    Article  CAS  PubMed  Google Scholar 

  18. Aachmann, F.L. et al. in Encyclopedia of Inorganic and Bioinorganic Chemistry (ed. Scott, R.A.) 111, 1–13 (Wiley, 2015).

    Google Scholar 

  19. Kjaergaard, C.H. et al. Spectroscopic and computational insight into the activation of O2 by the mononuclear Cu center in polysaccharide monooxygenases. Proc. Natl. Acad. Sci. USA 111, 8797–8802 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kim, S., Ståhlberg, J., Sandgren, M., Paton, R.S. & Beckham, G.T. Quantum mechanical calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl, oxygen-rebound mechanism. Proc. Natl. Acad. Sci. USA 111, 149–154 (2014).

    Article  CAS  PubMed  Google Scholar 

  21. Gao, J., Thomas, D.A., Sohn, C.H. & Beauchamp, J.L. Biomimetic reagents for the selective free radical and acid-base chemistry of glycans: application to glycan structure determination by mass spectrometry. J. Am. Chem. Soc. 135, 10684–10692 (2013).

    Article  CAS  PubMed  Google Scholar 

  22. Eriksson, K.-E., Pettersson, B. & Westermark, U. Oxidation: an important enzyme reaction in fungal degradation of cellulose. FEBS Lett. 49, 282–285 (1974).

    Article  CAS  PubMed  Google Scholar 

  23. Hemsworth, G.R. et al. The copper active site of CBM33 polysaccharide oxygenases. J. Am. Chem. Soc. 135, 6069–6077 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Li, X., Beeson, W.T. IV, Phillips, C.M., Marletta, M.A. & Cate, J.H.D. Structural basis for substrate targeting and catalysis by fungal polysaccharide monooxygenases. Structure 20, 1051–1061 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Agger, J.W. et al. Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation. Proc. Natl. Acad. Sci. USA 111, 6287–6292 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Borisova, A.S. et al. Structural and functional characterization of a lytic polysaccharide monooxygenase with broad substrate specificity. J. Biol. Chem. 290, 22955–22969 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Bennati-Granier, C. et al. Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina. Biotechnol. Biofuels 8, 90 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Loose, J.S.M., Forsberg, Z., Fraaije, M.W., Eijsink, V.G.H. & Vaaje-Kolstad, G. A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase. FEBS Lett. 588, 3435–3440 (2014).

    Article  CAS  PubMed  Google Scholar 

  29. Gudmundsson, M. et al. Structural and electronic snapshots during the transition from a Cu(II) to Cu(I) metal center of a lytic polysaccharide monooxygenase by X-ray photoreduction. J. Biol. Chem. 289, 18782–18792 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Asensio, J.L., Ardá, A., Cañada, F.J. & Jiménez-Barbero, J. Carbohydrate-aromatic interactions. Acc. Chem. Res. 46, 946–954 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Nishio, M. The CH/π hydrogen bond in chemistry. Conformation, supramolecules, optical resolution and interactions involving carbohydrates. Phys. Chem. Chem. Phys. 13, 13873–13900 (2011).

    Article  CAS  PubMed  Google Scholar 

  32. Davies, G.J., Planas, A. & Rovira, C. Conformational analyses of the reaction coordinate of glycosidases. Acc. Chem. Res. 45, 308–316 (2012).

    Article  CAS  PubMed  Google Scholar 

  33. Lee, J.Y. & Karlin, K.D. Elaboration of copper-oxygen mediated C-H activation chemistry in consideration of future fuel and feedstock generation. Curr. Opin. Chem. Biol. 25, 184–193 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Iwaizumi, M., Kudo, T. & Kita, S. Correlation between the hyperfine coupling constants of donor nitrogens and the structures of the first coordination sphere in copper complexes as studied by nitrogen-14 ENDOR spectroscopy. Inorg. Chem. 25, 1546–1550 (1986).

    Article  CAS  Google Scholar 

  35. Kim, D., Kim, N.H. & Kim, S.H. 34 GHz pulsed ENDOR characterization of the copper coordination of an amyloid-β peptide relevant to Alzheimer's disease. Angew. Chem. Int. Ed. Engl. 52, 1139–1142 (2013).

    Article  CAS  PubMed  Google Scholar 

  36. Aachmann, F.L., Sørlie, M., Skjåk-Bræk, G., Eijsink, V.G.H. & Vaaje-Kolstad, G. NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions. Proc. Natl. Acad. Sci. USA 109, 18779–18784 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Karlin, K.D. & Itoh, S. Copper-Oxygen Chemistry (Wiley, 2011).

  38. Donoghue, P.J. et al. Rapid C-H bond activation by a monocopper(III)-hydroxide complex. J. Am. Chem. Soc. 133, 17602–17605 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Wu, M. et al. Crystal structure and computational characterization of the lytic polysaccharide monooxygenase GH61D from the Basidiomycota fungus Phanerochaete chrysosporium. J. Biol. Chem. 288, 12828–12839 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Dhar, D. & Tolman, W.B. Hydrogen atom abstraction from hydrocarbons by a copper(III)-hydroxide complex. J. Am. Chem. Soc. 137, 1322–1329 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Singh, S.K. & Das, A. The n → π* interaction: a rapidly emerging non-covalent interaction. Phys. Chem. Chem. Phys. 17, 9596–9612 (2015).

    Article  CAS  PubMed  Google Scholar 

  42. Egli, M. & Gessner, R.V. Stereoelectronic effects of deoxyribose O4′ on DNA conformation. Proc. Natl. Acad. Sci. USA 92, 180–184 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Pavlakos, I. et al. Noncovalent lone pair···(No-π!)-heteroarene interactions: the Janus-faced hydroxy group. Angew. Chem. Int. Ed. Engl. 54, 8169–8174 (2015).

    Article  CAS  PubMed  Google Scholar 

  44. Roeser, D. et al. A general binding mechanism for all human sulfatases by the formylglycine-generating enzyme. Proc. Natl. Acad. Sci. USA 103, 81–86 (2006).

    Article  CAS  PubMed  Google Scholar 

  45. Spodsberg, N., Shaghasi, T., Sweeney, M., Xu, F. & Schnorr, K. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same. WO patent 2014066141 A3 (2014).

  46. Liu, Y. et al. Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction. Anal. Biochem. 267, 331–335 (1999).

    Article  CAS  PubMed  Google Scholar 

  47. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22–25 (2010).

    Article  CAS  PubMed  Google Scholar 

  49. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  50. Vagin, A.A. et al. REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr. D Biol. Crystallogr. 60, 2184–2195 (2004).

    Article  PubMed  Google Scholar 

  51. Gemperle, C., Aebli, G., Schweiger, A. & Ernst, R.R. Phase cycling in pulse EPR. J. Magn. Reson. 88, 241–256 (1990).

    CAS  Google Scholar 

Download references

Acknowledgements

We thank K. Rasmussen and R.M. Borup for experimental assistance, and MAXLAB, Sweden and the European Synchrotron Radiation Facility (ESRF), France, for synchrotron beam time and assistance. This work was supported by the UK Biotechnology and Biological Sciences Research Council (grant numbers BB/L000423 to P.D., G.J.D. and P.H.W., and BB/L021633/1 to G.J.D. and P.H.W.), Agence Française de l'Environnement et de la Maîtrise de l'Energie (grant number 1201C102 to B.H.), the Danish Council for Strategic Research (grant numbers 12-134923 to L.L.L. and 12-134922 to K.S.J.). Travel to synchrotrons was supported by the Danish Ministry of Higher Education and Science through the Instrument Center DANSCATT and the European Community's Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement 283570). L.M., S.F., S.C. and H.D. were supported by Institut de Chimie Moléculaire de Grenoble FR 2607, LabEx ARCANE (ANR-11-LABX-0003-01), the PolyNat Carnot Institute and the French Agence Nationale de la Recherche (PNRB2005-11).

Author information

Author notes

  1. Katja S Johansen

    Present address: Present address: Division of Industrial Biotechnology, Chalmers University of Technology, Göteborg, Sweden.,

Authors and Affiliations

  1. Department of Chemistry, University of Copenhagen, Copenhagen, Denmark

    Kristian E H Frandsen, Jens-Christian N Poulsen & Leila Lo Leggio

  2. Department of Biochemistry, University of Cambridge, Cambridge, UK

    Thomas J Simmons & Paul Dupree

  3. Department of Chemistry, University of York, York, UK

    Glyn R Hemsworth, Luisa Ciano, Esther M Johnston, Gideon J Davies & Paul H Walton

  4. Novozymes A/S, Bagsvaerd, Denmark

    Morten Tovborg, Katja S Johansen & Pernille von Freiesleben

  5. Centre de Recherches sur les Macromolecules Végétales (CERMAV), Université de Grenoble Alpes, Centre National de la Recherche Scientifique (CNRS), Grenoble, France

    Laurence Marmuse, Sébastien Fort, Sylvain Cottaz & Hugues Driguez

  6. Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS, Aix-Marseille Université, Marseille, France

    Bernard Henrissat & Nicolas Lenfant

  7. Institut National de la Recherche Agronomique (INRA), AFMB, Marseille, France

    Bernard Henrissat & Nicolas Lenfant

  8. Department of Biological Sciences, King Abdulaziz University, Jeddah, Saudi Arabia

    Bernard Henrissat

  9. Engineering and Physical Sciences Research Council (EPSRC) National EPR Facility, School of Chemistry and Photon Science Institute, University of Manchester, Manchester, UK

    Floriana Tuna & Amgalanbaatar Baldansuren

Authors
  1. Kristian E H Frandsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thomas J Simmons
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Paul Dupree
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jens-Christian N Poulsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Glyn R Hemsworth
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Luisa Ciano
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Esther M Johnston
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Morten Tovborg
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Katja S Johansen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Pernille von Freiesleben
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Laurence Marmuse
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Sébastien Fort
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Sylvain Cottaz
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Hugues Driguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Bernard Henrissat
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Nicolas Lenfant
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Floriana Tuna
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Amgalanbaatar Baldansuren
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Gideon J Davies
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Leila Lo Leggio
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Paul H Walton
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.E.H.F. crystallized protein, collected and analyzed crystallographic data, solved crystal structures and made structural figures and tables; P.D. and T.J.S. conceived the activity, oxidation state and MS experiments, and T.J.S. performed them; J.-C.N.P. crystallized protein and collected crystallographic data; G.R.H. designed and performed the FRET kinetics experiments; L.C. performed EPR experiments and simulations; E.M.J. performed EPR experiments; M.T. and K.S.J. oversaw and directed the work of P.v.F., who purified the recombinant enzymes; L.M., S.C., S.F. and H.D. conceived and performed the FRET substrate synthesis; B.H. and N.L. performed bioinformatics analyses and alignments; F.T. collected pulsed EPR data; A.B. collected and simulated pulsed EPR spectra; G.J.D. conceived the FRET kinetics study; L.L.L. conceived the crystallographic study, collected and analyzed crystallographic data and solved crystal structures. P.H.W. conceived the EPR study. P.H.W. and L.L.L. wrote the paper with contributions from all authors.

Corresponding authors

Correspondence to Leila Lo Leggio or Paul H Walton.

Ethics declarations

Competing interests

M.T. and P.v.F. are employees of Novozymes, a producer of enzymes for industrial use.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–6, Supplementary Notes 1–3 and Supplementary Figures 1–9. (PDF 1465 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Frandsen, K., Simmons, T., Dupree, P. et al. The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases. Nat Chem Biol 12, 298–303 (2016). https://doi.org/10.1038/nchembio.2029

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2029

This article is cited by

Search

Advanced 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