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.
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Protein Data Bank
Cragg, S.M. et al. Lignocellulose degradation mechanisms across the Tree of Life. Curr. Opin. Chem. Biol. 29, 108–119 (2015).
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).
Vaaje-Kolstad, G. et al. An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides. Science 330, 219–222 (2010).
Horn, S.J., Vaaje-Kolstad, G., Westereng, B. & Eijsink, V.G. Novel enzymes for the degradation of cellulose. Biotechnol. Biofuels 5, 45 (2012).
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).
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).
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).
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).
Walton, P.H. & Davies, G.J. On the catalytic mechanisms of lytic polysaccharide monooxygenases. Curr. Opin. Chem. Biol. 31, 195–207 (2016).
Kracher, D. et al. Extracellular electron transfer systems fuel cellulose oxidative degradation. Science 352, 1098–1101 (2016).
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).
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).
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).
Johansen, K.S. Discovery and industrial applications of lytic polysaccharide mono-oxygenases. Biochem. Soc. Trans. 44, 143–149 (2016).
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).
Frandsen, K.E.H. et al. The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases. Nat. Chem. Biol. 12, 298–303 (2016).
Solomon, E.I. et al. Copper active sites in biology. Chem. Rev. 114, 3659–3853 (2014).
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).
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).
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).
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).
Wang, C. et al. Evidence that the fosfomycin-producing epoxidase, HppE, is a non-heme-iron peroxidase. Science 342, 991–995 (2013).
Cannella, D. et al. Light-driven oxidation of polysaccharides by photosynthetic pigments and a metalloenzyme. Nat. Commun. 7, 11134 (2016).
Bissaro, B. et al. Fueling biomass-degrading oxidative enzymes by light-driven water oxidation. Green Chem. 18, 5357–5366 (2016).
Loose, J.S.M. et al. Activation of bacterial lytic polysaccharide monooxygenases with cellobiose dehydrogenase. Protein Sci. 25, 2175–2186 (2016).
Frommhagen, M. et al. Lytic polysaccharide monooxygenases from Myceliophthora thermophila C1 differ in substrate preference and reducing agent specificity. Biotechnol. Biofuels 9, 186 (2016).
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).
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).
Cirino, P.C. & Arnold, F.H. A self-sufficient peroxide-driven hydroxylation biocatalyst. Angew. Chem. Int. Ed. Engl. 42, 3299–3301 (2003).
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).
Mirica, L.M., Ottenwaelder, X. & Stack, T.D. Structure and spectroscopy of copper − dioxygen complexes. Chem. Rev. 104, 1013–1045 (2004).
Berlemont, R. Distribution and diversity of enzymes for polysaccharide degradation in fungi. Sci. Rep. 7, 222 (2017).
Hofrichter, M. & Ullrich, R. Oxidations catalyzed by fungal peroxygenases. Curr. Opin. Chem. Biol. 19, 116–125 (2014).
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).
Isaksen, T. et al. A C4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides. J. Biol. Chem. 289, 2632–2642 (2014).
Buettner, G.R. & Jurkiewicz, B.A. Catalytic metals, ascorbate and free radicals: combinations to avoid. Radiat. Res. 145, 532–541 (1996).
Boatright, W.L. Oxygen dependency of one-electron reactions generating ascorbate radicals and hydrogen peroxide from ascorbic acid. Food Chem. 196, 1361–1367 (2016).
Garajova, S. et al. Single-domain flavoenzymes trigger lytic polysaccharide monooxygenases for oxidative degradation of cellulose. Sci. Rep. 6, 28276 (2016).
Langston, J.A. et al. Oxidoreductive cellulose depolymerization by the enzymes cellobiose dehydrogenase and glycoside hydrolase 61. Appl. Environ. Microbiol. 77, 7007–7015 (2011).
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).
Mishra, S. & Imlay, J. Why do bacteria use so many enzymes to scavenge hydrogen peroxide? Arch. Biochem. Biophys. 525, 145–160 (2012).
Switala, J. & Loewen, P.C. Diversity of properties among catalases. Arch. Biochem. Biophys. 401, 145–154 (2002).
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).
Barbusinski, K. Fenton reaction - controversy concerning the chemistry. Ecol. Chem. Eng. S 16, 347–358 (2009).
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).
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).
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).
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).
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).
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).
Westereng, B. et al. Efficient separation of oxidized cello-oligosaccharides generated by cellulose degrading lytic polysaccharide monooxygenases. J. Chromatogr. A 1271, 144–152 (2013).
Zougman, A., Selby, P.J. & Banks, R.E. Suspension trapping (STrap) sample preparation method for bottom-up proteomics analysis. Proteomics 14, 1006–1010 (2014).
Chambers, M.C. et al. A cross-platform toolkit for mass spectrometry and proteomics. Nat. Biotechnol. 30, 918–920 (2012).
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.
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.
The authors declare no competing financial interests.
About this article
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). https://doi.org/10.1038/nchembio.2470
Biotechnology for Biofuels (2021)
Biotechnology for Biofuels (2021)
Heterologous expression of Phanerochaete chrysosporium cellobiose dehydrogenase in Trichoderma reesei
Microbial Cell Factories (2021)
In situ measurements of oxidation–reduction potential and hydrogen peroxide concentration as tools for revealing LPMO inactivation during enzymatic saccharification of cellulose
Biotechnology for Biofuels (2021)
The lytic polysaccharide monooxygenase CbpD promotes Pseudomonas aeruginosa virulence in systemic infection
Nature Communications (2021)