Discovery, biochemical and structural characterization of a new LPMO family with novel features raises parallels to cu-proteins of unrelated function

Session: 
S4.4 Polysaccharide processing for biofuels
Code: 
OL4.4.2
Location (hall): 
Fucose
Start/end time: 
Tuesday, July 2, 2019 - 12:00 to 12:15
Kristian
Frandsen

Kristian Frandsen1,2, Aurore Labourel1, Tobias Tanddrup2, Mireille Haon1, Sacha Grisel1, Marie-Noëlle Rosso1, Bernard Henrissat4,5, Francis Martin3, Jean-Guy Berrin1, Leila Lo Leggio2

1Biodiversité et Biotechnologie Fongiques, UMR1163, INRA, Aix-Marseille Université, Marseille, France, 2Department of Chemistry, University of Copenhagen, Copenhagen , Denmark, 3Interactions Arbres/Microorganismes, Laboratoire d’Excellence ARBRE, UMR1136, INRA, Université de Lorraine, Nancy, France, 4Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR7257, CNRS, Aix-Marseille Université, Marseille,  France , 5INRA, USC1408 AFMB, Marseille, France

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent [1] redox enzymes that oxidatively [2] modify carbohydrate biomass and play a key role in nature e.g. during fungal degradation of plant polysaccharides. The LPMO active site contains a copper atom coordinated by the “Histidine brace” motif (His brace, composed of an N-terminal histidine and a second histidine) highly conserved in members across all families. Currently, six LPMO families exist (AA9-AA11 and AA13-AA15 in the CAZy database [3]) and members are found in fungi, bacteria, viruses and recently in arthropod species (e.g insects) [4,5]. 

Recently we discovered a new LPMO family found in various lineages of both saprotrophic and ectomycorrhizal fungi. Members of this family invariably harbor a C-terminal glycosylphosphatidylinositol (GPI) anchor. Through transcriptomic analysis, we found that in some ectomycorrhizal fungi the genes encoding these new LPMO family members are upregulated during ectomycorrhizal- and fruiting body formation and immunolabelling experiments revealed that the corresponding LPMO proteins locate to the interface between fungal hyphae and tree rootlet cells, suggesting a role in the symbiosis-related cell wall remodelling. 

In this presentation, we focus on the structural characterization of one family member from the saprotroph basidiomycete fungus Laetisaria arvalis, with demonstrated LPMO activity on cellulose. We have determined the X-ray crystal structures of this new LPMO (in three different crystal forms) which show an antiparallel β-sheet topology typical of LPMOs. However, LaLPMO has a diminished active site surface compared to other LPMOs. In addition, all structures show the copper coordinated by a canonical His brace, but also an unusual Asp ligand. Bioinformatic analyses show that the majority of the sequences in the family have a similar Asp ligand, but that a subgroup of sequence appear to have a third His instead of the Asp ligand. These active site arrangements are reminiscent both of an unrelated protein CopC likely involved in copper homeostasis [6], but also of particulate methane monooxygenases (pMMOs) which has a monocopper site with three His ligands [7]. 

The results from our work expand the known biological and structural diversity of LPMOs and challenges the current view on their functional roles.

Acknowlegements

KF was funded through a Postdoc Fellowship from the Carlsberg Foundation (grant n°CF16-0673 and n°CF17-0533) and an AgreenSkills+ fellowship (Marie-Curie FP7 COFUND People Programme, grant agreement n°609398). AL was funded by a Marie Curie Individual Fellowship within the Horizon 2020 Research and Innovation Framework Programme (748758). We thank Katja S. Johansen (KSJ) for helpful discussions. KF, TT, KSJ and LLL are members of the HOPE project (https://ign.ku.dk/hope/) funded by the Novo Nordisk Foundation (grant NNF17SA0027704).

References: 
  1. Quinlan, R.J. et al. (2011). Proc. Natl. Acad. Sci. U. S. A 108 (37) 15079-15084. 
  2. Vaaje-Kolstad, G. et al. (2010). Science 330:219. 
  3. Levasseur, A. et al. (2013) Biotechnol. Biofuels 6.

  4. Tandrup, T. et al. (2018) Biochem. Soc. Trans. 46(6) 1431-47.

  5. Johansen, K.S. (2016) Trends Plant Sci. 21(11) 926-36.

  6. Lawton, T.J. et al. (2016). Biochemistry. 55(15):2278-90.

  7. Cao, L. et al. (2018) Angew. Chem. 57(1):162-6

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