title: Interactions of a fungal lytic polysaccharide monooxygenase with β-glucan substrates and cellobiose dehydrogenase.
abstract: Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds using molecular oxygen and an external electron donor. We have used NMR and isothermal titration calorimetry (ITC) to study the interactions of a broad-specificity fungal LPMO, NcLPMO9C, with various substrates and with cellobiose dehydrogenase (CDH), a known natural supplier of electrons. The NMR studies revealed interactions with cellohexaose that center around the copper site. NMR studies with xyloglucans, i.e., branched β-glucans, showed an extended binding surface compared with cellohexaose, whereas ITC experiments showed slightly higher affinity and a different thermodynamic signature of binding. The ITC data also showed that although the copper ion alone hardly contributes to affinity, substrate binding is enhanced for metal-loaded enzymes that are supplied with cyanide, a mimic of O2 (-) Studies with CDH and its isolated heme b cytochrome domain unambiguously showed that the cytochrome domain of CDH interacts with the copper site of the LPMO and that substrate binding precludes interaction with CDH. Apart from providing insights into enzyme-substrate interactions in LPMOs, the present observations shed new light on possible mechanisms for electron supply during LPMO action.
authors.affiliation: Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway;
authors.forename: Gaston
authors.initials: G
authors.lastname: Courtade
authors.affiliation: Department of Chemistry and Bioscience, Aalborg University, DK-9220 Aalborg Ø, Denmark;
authors.forename: Reinhard
authors.identifier: 0000-0001-6942-5056
authors.initials: R
authors.lastname: Wimmer
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, N-1432 Ås, Norway;
authors.forename: Åsmund K
authors.initials: ÅK
authors.lastname: Røhr
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, Vienna Institute of Biotechnology, University of Natural Resources and Life Sciences, Vienna A-1190, Austria;
authors.forename: Marita
authors.initials: M
authors.lastname: Preims
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, Vienna Institute of Biotechnology, University of Natural Resources and Life Sciences, Vienna A-1190, Austria;
authors.forename: Alfons K G
authors.initials: AK
authors.lastname: Felice
authors.affiliation: Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Dimarogona
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, N-1432 Ås, Norway;
authors.forename: Gustav
authors.initials: G
authors.lastname: Vaaje-Kolstad
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, N-1432 Ås, Norway;
authors.forename: Morten
authors.initials: M
authors.lastname: Sørlie
authors.affiliation: Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Sandgren
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, Vienna Institute of Biotechnology, University of Natural Resources and Life Sciences, Vienna A-1190, Austria;
authors.forename: Roland
authors.initials: R
authors.lastname: Ludwig
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, N-1432 Ås, Norway; finn.l.aachmann@ntnu.no vincent.eijsink@nmbu.no.
authors.forename: Vincent G H
authors.initials: VG
authors.lastname: Eijsink
authors.affiliation: Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway; finn.l.aachmann@ntnu.no vincent.eijsink@nmbu.no.
authors.forename: Finn Lillelund
authors.identifier: 0000-0003-1613-4663
authors.initials: FL
authors.lastname: Aachmann
mesh_terms: D001665:Binding Sites; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry* / Q000235:genetics; D003300:Copper / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D003300:Copper; D006899:Mixed Function Oxygenases; D002237:Carbohydrate Dehydrogenases; C019859:cellobiose-quinone oxidoreductase
keywords: LPMO; cellobiose dehydrogenase; cellulose; lytic polysaccharide monooxygenase; xyloglucan