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The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases

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.

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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.

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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).

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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.

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Correspondence to Leila Lo Leggio or Paul H Walton.

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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. (PDF 1465 kb)

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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

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