The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases

Journal name:
Nature Chemical Biology
Year published:
Published online


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.

At a glance


  1. LPMO activity and active site.
    Figure 1: LPMO activity and active site.

    (a) Oxidative cleavage of a polysaccharide chain carried out by LPMOs, showing different routes of oxidation carried out by LPMO subgroups. (b) The histidine brace active site and proposed first step in the reaction with oxygen19.

  2. Ls(AA9)A and Ta(AA9)A activity on insoluble and soluble cellulose.
    Figure 2: Ls(AA9)A and Ta(AA9)A activity on insoluble and soluble cellulose.

    (a) PACE of Ls(AA9)A and Ta(AA9)A activities on PASC, with (+) or without (−) ascorbate (performed in triplicate). (b) Schematic (top) and Michaelis–Menten plot (bottom) of cleavage of FRET substrate by Ls(AA9)A. Error bars represent s.d. of triplicate measurements.

  3. Structural views of Ls(AA9)A.
    Figure 3: Structural views of Ls(AA9)A.

    (a) Ribbon view of Ls(AA9)A with Cu(I) (orange sphere). (b) Electron density of active site obtained with low X-ray dose; Cu(II) is shown as a blue sphere. Me-H1, N-terminal methylated histidine; (c) Ls(AA9)A (left) and Ta(AA9)A (right) surfaces. Ls(AA9)A-G3 (low dose) with Tyr203 is shown in red. Ta(AA9)A (PDB 3ZUD) with Tyr24 and Tyr212 are shown in red with superimposed structure of the G3 from Ls(AA9)A-G3. The N-terminal me-H1 is shown in blue. (d) Principal protein contacts between G3 and the binding surface of Ls(AA9)A in Ls(AA9)A-G3 structure with high-dose X-rays. Cu(I) is shown as an orange sphere. (e) Electron density of Ls(AA9)A-G3 with low X-ray dose. Cu(II) is shown as a blue sphere. H2Oax and H2Oeq represent the water ligands on the axial and equatorial positions, respectively.

  4. Structure of Ls(AA9)A around active site before and after binding of G3 (R = H).
    Figure 4: Structure of Ls(AA9)A around active site before and after binding of G3 (R = H).

    'Pocket' water molecule is shown in red.

  5. EPR spectra of Ls(AA9)A at 150 K.
    Figure 5: EPR spectra of Ls(AA9)A at 150 K.

    (a) X-band cw EPR spectra in low-chloride conditions for Ls(AA9)A-G6 (top) and Ls(AA9)A with no oligosaccharide (bottom). (b) X-band cw EPR spectra in high-chloride conditions for Ls(AA9)A-G6 (top) and Ls(AA9)A with no oligosaccharide (bottom). (c) Contour presentation of the 1H-HYSCORE spectrum of Ls(AA9)A-G6 in the presence of chloride ions (magnetic field 343.6 mT, time between first and second pulse τ = 136 ns, microwave frequency 9.7 GHz). The antidiagonal crosspeaks are assigned to anisotropic protons (1H and 2H). *, signals from copper site where water was the exogenous ligand (other signals were from species with chloride as exogenous ligand). X-band cw EPR measurements were repeated in at least triplicate.


8 compounds View all compounds
  1. Hepta-O-acetyl α-lactosyl fluoride
    Compound 1 Hepta-O-acetyl α-lactosyl fluoride
  2. 2,3,4-Tri-O-acetyl-6-O-levulinyl-β-D-galactopyranosyl-(1,4)-2,3,6-tri-O-acetyl-α-D-glucopyranosyl fluoride
    Compound 2 2,3,4-Tri-O-acetyl-6-O-levulinyl-β-D-galactopyranosyl-(1,4)-2,3,6-tri-O-acetyl-α-D-glucopyranosyl fluoride
  3. 2,3,4-Tri-O-acetyl-6-O-methanesulfonyl-β-D-galactopyranosyl-(1,4)-2,3,6-tri-O-acetyl-α-D-glucopyranosyl fluoride
    Compound 3 2,3,4-Tri-O-acetyl-6-O-methanesulfonyl-β-D-galactopyranosyl-(1,4)-2,3,6-tri-O-acetyl-α-D-glucopyranosyl fluoride
  4. 2,3,4-Tri-O-acetyl-6-O-azido-6-O-deoxy-β-D-galactopyranosyl-(1,4)-2,3,6-tri-O-acetyl-α-D-glucopyranosyl fluoride
    Compound 4 2,3,4-Tri-O-acetyl-6-O-azido-6-O-deoxy-β-D-galactopyranosyl-(1,4)-2,3,6-tri-O-acetyl-α-D-glucopyranosyl fluoride
  5. 6-O-azido-6-O-deoxy-β-D-galactopyranosyl-(1,4)-α-D-glucopyranosyl fluoride
    Compound 5 6-O-azido-6-O-deoxy-β-D-galactopyranosyl-(1,4)-α-D-glucopyranosyl fluoride
  6. Sodium N-[2-N[(S-β-D-glucopyranosyl-(1,4)-β-D-glucopyranosyl-2-thioacetyl]aminoethyl]-1-naphthylamine-5-sulfonate
    Compound 6 Sodium N-[2-N[(S-β-D-glucopyranosyl-(1,4)-β-D-glucopyranosyl-2-thioacetyl]aminoethyl]-1-naphthylamine-5-sulfonate
  7. Sodium N-[2-N[(S-(6-azido-6-deoxy-β-D-galactopyranosyl-(1,4)-β-D-glucopyranosyl-(1,4)-β-D-glucopyranosyl)-(1,4)-β-D-glucopyranosyl)-2-thioacetyl]aminoethyl]-1-naphthylamine-5-sulfonate
    Compound 7 Sodium N-[2-N[(S-(6-azido-6-deoxy-β-D-galactopyranosyl-(1,4)-β-D-glucopyranosyl-(1,4)-β-D-glucopyranosyl)-(1,4)-β-D-glucopyranosyl)-2-thioacetyl]aminoethyl]-1-naphthylamine-5-sulfonate
  8. Sodium N-[2-N[(S-(6-deoxy-6-(4-((4-(dimethylamino)phenyl)azo)phenylthioureido-β-D-galactopyranosyl-(1,4)-β-D-glucopyranosyl-(1,4)-β-D-glucopyranosyl)-(1,4)-β-D-glucopyranosyl)-2-thioacetyl]aminoethyl]-1-naphthylamine-5-sulfonate
    Compound 8 Sodium N-[2-N[(S-(6-deoxy-6-(4-((4-(dimethylamino)phenyl)azo)phenylthioureido-β-D-galactopyranosyl-(1,4)-β-D-glucopyranosyl-(1,4)-β-D-glucopyranosyl)-(1,4)-β-D-glucopyranosyl)-2-thioacetyl]aminoethyl]-1-naphthylamine-5-sulfonate

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Referenced accessions


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Author information

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

    • Katja S Johansen


  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


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.

Competing financial interests

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

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    Supplementary Results, Supplementary Tables 1–6, Supplementary Notes 1–3 and Supplementary Figures 1–9.

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