Skip to main content

Thank you for visiting 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.

Oxidative cleavage of polysaccharides by monocopper enzymes depends on H2O2


Enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) play an important role in the conversion of recalcitrant polysaccharides, but their mode of action has remained largely enigmatic. It is generally believed that catalysis by LPMOs requires molecular oxygen and a reductant that delivers two electrons per catalytic cycle. Using enzyme assays, mass spectrometry and experiments with labeled oxygen atoms, we show here that H2O2, rather than O2, is the preferred co-substrate of LPMOs. By controlling H2O2 supply, stable reaction kinetics are achieved, the LPMOs work in the absence of O2, and the reductant is consumed in priming rather than in stoichiometric amounts. The use of H2O2 by a monocopper enzyme that is otherwise cofactor-free offers new perspectives regarding the mode of action of copper enzymes. Furthermore, these findings have implications for the enzymatic conversion of biomass in Nature and in industrial biorefining.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Probing the role of H2O2 in LPMO catalysis.
Figure 2: Location of oxidative modifications in ScLPMO10C.
Figure 3: Effect of the H2O2 feeding rate on LPMO activity in a commercial cellulolytic enzyme cocktail, reductant consumption and glucose release during saccharification of crystalline cellulose.
Figure 4: Putative LPMO-guided H2O2 splitting mechanism for enzymatic oxidative cleavage of polysaccharides.

Accession codes


Protein Data Bank


  1. 1

    Cragg, S.M. et al. Lignocellulose degradation mechanisms across the Tree of Life. Curr. Opin. Chem. Biol. 29, 108–119 (2015).

    CAS  Article  Google Scholar 

  2. 2

    Arantes, V. & Goodell, B. in Deterioration and Protection of Sustainable Biomaterials. ACS Symposium Series Vol. 1158 (eds. Shultz, P., Goodell, B. & Nicholas D.D.) Ch. 1 (American Chemical Society, 2014).

  3. 3

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

    CAS  Article  PubMed  Google Scholar 

  4. 4

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5

    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).

    CAS  Article  PubMed  Google Scholar 

  6. 6

    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).

    CAS  Article  PubMed  Google Scholar 

  7. 7

    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).

    CAS  Article  Google Scholar 

  8. 8

    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).

    CAS  Article  Google Scholar 

  9. 9

    Walton, P.H. & Davies, G.J. On the catalytic mechanisms of lytic polysaccharide monooxygenases. Curr. Opin. Chem. Biol. 31, 195–207 (2016).

    CAS  Article  Google Scholar 

  10. 10

    Kracher, D. et al. Extracellular electron transfer systems fuel cellulose oxidative degradation. Science 352, 1098–1101 (2016).

    CAS  Article  Google Scholar 

  11. 11

    Kirn, T.J., Jude, B.A. & Taylor, R.K. A colonization factor links Vibrio cholerae environmental survival and human infection. Nature 438, 863–866 (2005).

    CAS  Article  Google Scholar 

  12. 12

    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).

    CAS  Article  Google Scholar 

  13. 13

    Hu, J. et al. Substrate factors that influence the synergistic interaction of AA9 and cellulases during the enzymatic hydrolysis of biomass. Energy Environ. Sci. 7, 2308–2315 (2014).

    CAS  Article  Google Scholar 

  14. 14

    Johansen, K.S. Discovery and industrial applications of lytic polysaccharide mono-oxygenases. Biochem. Soc. Trans. 44, 143–149 (2016).

    CAS  Article  Google Scholar 

  15. 15

    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).

    CAS  Article  Google Scholar 

  16. 16

    Frandsen, K.E.H. et al. The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases. Nat. Chem. Biol. 12, 298–303 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17

    Solomon, E.I. et al. Copper active sites in biology. Chem. Rev. 114, 3659–3853 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18

    Torres Pazmiño, D.E., Winkler, M., Glieder, A. & Fraaije, M.W. Monooxygenases as biocatalysts: classification, mechanistic aspects and biotechnological applications. J. Biotechnol. 146, 9–24 (2010).

    Article  Google Scholar 

  19. 19

    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).

    CAS  Article  Google Scholar 

  20. 20

    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 

  21. 21

    Kittl, R., Kracher, D., Burgstaller, D., Haltrich, D. & Ludwig, R. Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay. Biotechnol. Biofuels 5, 79 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22

    Wang, C. et al. Evidence that the fosfomycin-producing epoxidase, HppE, is a non-heme-iron peroxidase. Science 342, 991–995 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23

    Cannella, D. et al. Light-driven oxidation of polysaccharides by photosynthetic pigments and a metalloenzyme. Nat. Commun. 7, 11134 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24

    Bissaro, B. et al. Fueling biomass-degrading oxidative enzymes by light-driven water oxidation. Green Chem. 18, 5357–5366 (2016).

    CAS  Article  Google Scholar 

  25. 25

    Loose, J.S.M. et al. Activation of bacterial lytic polysaccharide monooxygenases with cellobiose dehydrogenase. Protein Sci. 25, 2175–2186 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26

    Frommhagen, M. et al. Lytic polysaccharide monooxygenases from Myceliophthora thermophila C1 differ in substrate preference and reducing agent specificity. Biotechnol. Biofuels 9, 186 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  27. 27

    Warren, J.J., Ener, M.E., Vlček, A. Jr., Winkler, J.R. & Gray, H.B. Electron hopping through proteins. Coord. Chem. Rev. 256, 2478–2487 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28

    Forsberg, Z. et al. Structural and functional characterization of a conserved pair of bacterial cellulose-oxidizing lytic polysaccharide monooxygenases. Proc. Natl. Acad. Sci. USA 111, 8446–8451 (2014).

    CAS  Article  Google Scholar 

  29. 29

    Cirino, P.C. & Arnold, F.H. A self-sufficient peroxide-driven hydroxylation biocatalyst. Angew. Chem. Int. Ed. Engl. 42, 3299–3301 (2003).

    CAS  Article  Google Scholar 

  30. 30

    Hrycay, E.G. & Bandiera, S.M. Monooxygenase, peroxidase and peroxygenase properties and reaction mechanisms of cytochrome P450 enzymes. Adv. Exp. Med. Biol. 851, 1–61 (2015).

    CAS  Article  Google Scholar 

  31. 31

    Mirica, L.M., Ottenwaelder, X. & Stack, T.D. Structure and spectroscopy of copper − dioxygen complexes. Chem. Rev. 104, 1013–1045 (2004).

    CAS  Article  Google Scholar 

  32. 32

    Berlemont, R. Distribution and diversity of enzymes for polysaccharide degradation in fungi. Sci. Rep. 7, 222 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  33. 33

    Hofrichter, M. & Ullrich, R. Oxidations catalyzed by fungal peroxygenases. Curr. Opin. Chem. Biol. 19, 116–125 (2014).

    CAS  Article  Google Scholar 

  34. 34

    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).

    CAS  Article  Google Scholar 

  35. 35

    Isaksen, T. et al. A C4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides. J. Biol. Chem. 289, 2632–2642 (2014).

    CAS  Article  Google Scholar 

  36. 36

    Buettner, G.R. & Jurkiewicz, B.A. Catalytic metals, ascorbate and free radicals: combinations to avoid. Radiat. Res. 145, 532–541 (1996).

    CAS  Article  Google Scholar 

  37. 37

    Boatright, W.L. Oxygen dependency of one-electron reactions generating ascorbate radicals and hydrogen peroxide from ascorbic acid. Food Chem. 196, 1361–1367 (2016).

    CAS  Article  Google Scholar 

  38. 38

    Garajova, S. et al. Single-domain flavoenzymes trigger lytic polysaccharide monooxygenases for oxidative degradation of cellulose. Sci. Rep. 6, 28276 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 39

    Langston, J.A. et al. Oxidoreductive cellulose depolymerization by the enzymes cellobiose dehydrogenase and glycoside hydrolase 61. Appl. Environ. Microbiol. 77, 7007–7015 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40

    Scott, B.R., Huang, H.Z., Frickman, J., Halvorsen, R. & Johansen, K.S. Catalase improves saccharification of lignocellulose by reducing lytic polysaccharide monooxygenase-associated enzyme inactivation. Biotechnol. Lett. 38, 425–434 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41

    Mishra, S. & Imlay, J. Why do bacteria use so many enzymes to scavenge hydrogen peroxide? Arch. Biochem. Biophys. 525, 145–160 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42

    Switala, J. & Loewen, P.C. Diversity of properties among catalases. Arch. Biochem. Biophys. 401, 145–154 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43

    Beeson, W.T., Phillips, C.M., Cate, J.H.D. & Marletta, M.A. Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases. J. Am. Chem. Soc. 134, 890–892 (2012).

    CAS  Article  PubMed  Google Scholar 

  44. 44

    Barbusinski, K. Fenton reaction - controversy concerning the chemistry. Ecol. Chem. Eng. S 16, 347–358 (2009).

    CAS  Google Scholar 

  45. 45

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. 46

    Eibinger, M. et al. Cellulose surface degradation by a lytic polysaccharide monooxygenase and its effect on cellulase hydrolytic efficiency. J. Biol. Chem. 289, 35929–35938 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47

    Zámocký, M. et al. Cloning, sequence analysis and heterologous expression in Pichia pastoris of a gene encoding a thermostable cellobiose dehydrogenase from Myriococcum thermophilum. Protein Expr. Purif. 59, 258–265 (2008).

    Article  Google Scholar 

  48. 48

    Vaaje-Kolstad, G., Horn, S.J., van Aalten, D.M.F., Synstad, B. & Eijsink, V.G.H. The non-catalytic chitin-binding protein CBP21 from Serratia marcescens is essential for chitin degradation. J. Biol. Chem. 280, 28492–28497 (2005).

    CAS  Article  Google Scholar 

  49. 49

    Westereng, B. et al. The putative endoglucanase PcGH61D from Phanerochaete chrysosporium is a metal-dependent oxidative enzyme that cleaves cellulose. PLoS One 6, e27807 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. 50

    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 

  51. 51

    Westereng, B. et al. Efficient separation of oxidized cello-oligosaccharides generated by cellulose degrading lytic polysaccharide monooxygenases. J. Chromatogr. A 1271, 144–152 (2013).

    CAS  Article  Google Scholar 

  52. 52

    Zougman, A., Selby, P.J. & Banks, R.E. Suspension trapping (STrap) sample preparation method for bottom-up proteomics analysis. Proteomics 14, 1006–1010 (2014).

    CAS  Article  Google Scholar 

  53. 53

    Chambers, M.C. et al. A cross-platform toolkit for mass spectrometry and proteomics. Nat. Biotechnol. 30, 918–920 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references


We thank B. Westereng at Norwegian University of Life Sciences (NMBU), Ås and M. Sandgren at the Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden for providing a sample of a purified recombinant fungal AA9 (PcLPMO9D). B.B. has received the support of the EU in the framework of the Marie-Curie FP7 COFUND People Programme, through the award of an AgreenSkills fellowship (under grant agreement n° 267196). The postdoctoral fellowship of B.B. was also supported by the French Institut National de la Recherche Agronomique (INRA) [CJS]. This work was also supported by the Research Council of Norway through grants 214613, 240967, 243950 and 249865, and by the Vista programme of The Norwegian Academy of Science and Letters through grant 6510.

Authors contributions

B.B. designed, performed and analyzed most of the experiments. M.S. performed the proteomics experiments. G.M. and P.C. performed the bioreactor saccharification experiments. Z.F. and G.V.-K. provided enzymes. Å.K.R. and S.J.H. contributed to data analysis and experimental design. B.B. and V.G.H.E. supervised the study and wrote the manuscript. All authors participated in critical analysis of the data and finalizing the manuscript.

Author information



Corresponding authors

Correspondence to Bastien Bissaro or Vincent G H Eijsink.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results and Supplementary Figures 1–19. (PDF 4297 kb)

Reporting Summary (PDF 135 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Bissaro, B., Røhr, Å., Müller, G. et al. Oxidative cleavage of polysaccharides by monocopper enzymes depends on H2O2. Nat Chem Biol 13, 1123–1128 (2017).

Download citation

Further reading


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