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Oxidative cleavage of polysaccharides by monocopper enzymes depends on H2O2

Nature Chemical Biology volume 13, pages 1123–1128 (2017)Cite this article

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Abstract

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.

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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.

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Acknowledgements

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.

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  1. Institut National de la Recherche Agronomique (INRA), UMR792, Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France

    Bastien Bissaro

  2. Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), Ås, Norway

    Bastien Bissaro, Åsmund K Røhr, Gerdt Müller, Piotr Chylenski, Morten Skaugen, Zarah Forsberg, Svein J Horn, Gustav Vaaje-Kolstad & Vincent G H Eijsink

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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

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