title: The Contribution of Non-catalytic Carbohydrate Binding Modules to the Activity of Lytic Polysaccharide Monooxygenases.
abstract: Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharide monooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases,CfLPMO10 andTbLPMO10 fromCellulomonas fimiandThermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducingCtCBM3a, from theClostridium thermocellumcellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact ofCtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM fromCfLPMO10 or the introduction of a family 10 CBM fromCellvibrio japonicusLPMO10B intoTbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations.
authors.affiliation: From the Institute for Cell and Molecular Biosciences, The Medical School Newcastle University, Newcastle upon Tyne NE2 4HH and.
authors.forename: Lucy I
authors.initials: LI
authors.lastname: Crouch
authors.affiliation: From the Institute for Cell and Molecular Biosciences, The Medical School Newcastle University, Newcastle upon Tyne NE2 4HH and.
authors.forename: Aurore
authors.initials: A
authors.lastname: Labourel
authors.affiliation: the York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, United Kingdom.
authors.forename: Paul H
authors.initials: PH
authors.lastname: Walton
authors.affiliation: the York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, United Kingdom.
authors.forename: Gideon J
authors.initials: GJ
authors.lastname: Davies
authors.affiliation: From the Institute for Cell and Molecular Biosciences, The Medical School Newcastle University, Newcastle upon Tyne NE2 4HH and harry.gilbert@ncl.ac.uk.
authors.forename: Harry J
authors.initials: HJ
authors.lastname: Gilbert
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D040143:Cellulomonas / Q000201:enzymology* / Q000235:genetics; D020633:Cellvibrio / Q000201:enzymology* / Q000235:genetics; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D011135:Polysaccharides, Bacterial / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; C080369:CipA protein, Clostridium; D008565:Membrane Proteins; D011135:Polysaccharides, Bacterial; D006899:Mixed Function Oxygenases
keywords: carbohydrate binding modules; carbohydrate-binding protein; cellulases; enzyme; glycoside hydrolase; lignocellulose degradation; lytic polysaccharide monooxygenases; plant cell wall; protein engineering