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ABSTRACT Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in the oxidative degradation of various biopolymers such as cellulose and chitin.

While hunting for new LPMOs, we identified a new family of proteins, defined here as X325, in various fungal lineages. The three-dimensional structure of X325 revealed an overall LPMO fold

and a His brace with an additional Asp ligand to Cu(II). Although LPMO-type activity of X325 members was initially expected, we demonstrated that X325 members do not perform oxidative

cleavage of polysaccharides, establishing that X325s are not LPMOs. Investigations of the biological role of X325 in the ectomycorrhizal fungus _Laccaria bicolor_ revealed exposure of the

X325 protein at the interface between fungal hyphae and tree rootlet cells. Our results provide insights into a family of copper-containing proteins, which is widespread in the fungal

kingdom and is evolutionarily related to LPMOs, but has diverged to biological functions other than polysaccharide degradation. Access through your institution Buy or subscribe This is a

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* Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS INSIGHTS INTO PECULIAR FUNGAL LPMO FAMILY MEMBERS HOLDING

A SHORT C-TERMINAL SEQUENCE REMINISCENT OF PHOSPHATE BINDING MOTIFS Article Open access 18 July 2023 STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE CATALYTIC DOMAIN OF A CELL-WALL

ANCHORED BACTERIAL LYTIC POLYSACCHARIDE MONOOXYGENASE FROM _STREPTOMYCES COELICOLOR_ Article Open access 01 April 2023 FUNCTIONAL CHARACTERIZATION OF A LYTIC POLYSACCHARIDE MONOOXYGENASE

FROM _SCHIZOPHYLLUM COMMUNE_ THAT DEGRADES NON-CRYSTALLINE SUBSTRATES Article Open access 13 October 2023 DATA AVAILABILITY LaX325 nucleotide sequence was deposited in GenBank under

accession number MK088083. The X-ray structures of LaX325 were deposited in the Protein Data Bank under accession numbers 6IBH, 6IBI and 6IBJ. Raw EPR data are available on request through

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ACKNOWLEDGEMENTS A.L. was funded by a Marie Curie Individual Fellowship within the Horizon 2020 Research and Innovation Framework Programme (748758). The Danish Ministry of Higher Education

and Science through the Instrument Center DANSCATT funded travel to synchrotrons. We would also like to acknowledge MAX-IV, Lund, Sweden, the ESRF, Grenoble, France and DESY, Hamburg,

Germany for synchrotron beamtime and related assistance. We thank J.-C. Poulsen for technical assistance; A. Kohler and D. Thiele for helpful discussions; F. Chaspoul for assistance with

ICP-MS analyses and R. Balestrini (Institute for Sustainable Plant Protection, Italy) for providing gold-labeled WGA lectin. L.L.L. and T.T. thank the Novo Nordisk Foundation for funding

through grant NF17SA0027704. K.E.H.F. thanks the Carlsberg Foundation through an Internationalization Postdoc Fellowship (grants CF16-0673 and CF17-0533) and the EU, framework of the Marie

Curie FP7 COFUND People Programme (AgreenSkills+ fellowship 609398) for financial support. L.L.L., T.T. and K.E.H.F. are members of ISBUC Integrative Structural Biology at the University of

Copenhagen (https://isbuc.ku.dk/). K.S.J. thanks the Novo Nordisk Foundation for funding through grant NNF17SA0027704. P.H.W. and L.C. thank the UK Biotechnology and Biological Sciences

Research Council (BB/L021633/1) for funding. F.M. is funded by the French National Research Agency through the Laboratory of Excellence Advanced Research on the Biology of Tree and Forest

Ecosystems (grant ANR-11-LABX 0002 01). A.Z. thanks the French Embassy in Greece, together with the French Ministry of Higher Education, Research and Innovation, for the scholarship ‘Séjour

scientifique de haut niveau’ (SSHN). The electron microscopy experiments were performed on the PiCSL-FBI core facility (IBDM, Marseille), member of the France-BioImaging national research

infrastructure (ANR-10-INBS-04). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * INRA, Biodiversité et Biotechnologie Fongiques (BBF), UMR1163, Aix Marseille Université, Marseille, France

Aurore Labourel, Kristian E. H. Frandsen, Sacha Grisel, Mireille Haon, David Navarro, Marie-Noëlle Rosso, Bastien Bissaro, Anastasia Zerva & Jean-Guy Berrin * Biological Chemistry

Section, Department of Chemistry, University of Copenhagen, Copenhagen, Denmark Kristian E. H. Frandsen, Tobias Tandrup & Leila Lo Leggio * INRA, UMR1136, Interactions

Arbres/Microorganismes, Laboratoire d’Excellence ARBRE, Centre INRA-Lorraine, Université de Lorraine, Champenoux, France Feng Zhang & Francis Martin * CNRS, IBDM, Aix Marseille

Université, Marseille, France Nicolas Brouilly * Department of Chemistry, University of York, York, UK Luisa Ciano & Paul H. Walton * INRA, Unité de Recherche Biopolymères Interactions

Assemblages (BIA), Nantes, France David Ropartz & Mathieu Fanuel * Department of Geosciences and Natural Resource Management, University of Copenhagen, Copenhagen, Denmark Katja S.

Johansen * Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Zografou Campus, Athens, Greece Anastasia Zerva * INRA, USC1408 Architecture et

Fonction des Macromolécules Biologiques (AFMB), Marseille, France Bernard Henrissat * Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS, Aix Marseille Université,

Marseille, France Bernard Henrissat * Department of Biological Sciences, King Abdulaziz University, Jeddah, Saudi Arabia Bernard Henrissat Authors * Aurore Labourel View author publications

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Sacha Grisel View author publications You can also search for this author inPubMed Google Scholar * Mireille Haon View author publications You can also search for this author inPubMed Google

Scholar * Luisa Ciano View author publications You can also search for this author inPubMed Google Scholar * David Ropartz View author publications You can also search for this author

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can also search for this author inPubMed Google Scholar * Tobias Tandrup View author publications You can also search for this author inPubMed Google Scholar * Bastien Bissaro View author

publications You can also search for this author inPubMed Google Scholar * Katja S. Johansen View author publications You can also search for this author inPubMed Google Scholar * Anastasia

Zerva View author publications You can also search for this author inPubMed Google Scholar * Paul H. Walton View author publications You can also search for this author inPubMed Google

Scholar * Bernard Henrissat View author publications You can also search for this author inPubMed Google Scholar * Leila Lo Leggio View author publications You can also search for this

author inPubMed Google Scholar * Jean-Guy Berrin View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS A.L., D.N. and M.-N.R. identified the new

proteins. A.L. and B.H. performed bioinformatic analyses. A.L., S.G. and M.H. performed recombinant protein production and purification. T.T. and K.E.H.F. crystallized LaX325, determined and

analyzed the X-ray crystal structure. T.T. collected X-ray data. K.E.H.F. made relevant figures and tables. L.L.L. directed the crystallographic studies. K.E.H.F., K.S.J. and L.L.L.

analyzed the structure. K.E.H.F. and L.L.L. drafted relevant parts of the manuscript. A.L., B.B. and A.Z. performed enzyme assays. M.F. and D.R. performed mass spectrometry analyses. L.C.

and P.H.W. conceived and carried out the EPR study. A.L. and F.Z. performed confocal microscopy under the supervision of F.M. A.L. and N.B. performed transmission electron microscopy.

J.-G.B. coordinated the work. A.L. and J.-G.B. organized the data and drafted the manuscript. All authors made comments on the manuscript and approved the final version. CORRESPONDING AUTHOR

Correspondence to Jean-Guy Berrin. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains

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Figs. 1–9. REPORTING SUMMARY RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Labourel, A., Frandsen, K.E.H., Zhang, F. _et al._ A fungal family of lytic

polysaccharide monooxygenase-like copper proteins. _Nat Chem Biol_ 16, 345–350 (2020). https://doi.org/10.1038/s41589-019-0438-8 Download citation * Received: 09 January 2019 * Accepted: 22

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