title: Three-dimensional structures of two heavily N-glycosylated Aspergillus sp. family GH3 β-D-glucosidases.
abstract: The industrial conversion of cellulosic plant biomass into useful products such as biofuels is a major societal goal. These technologies harness diverse plant degrading enzymes, classical exo- and endo-acting cellulases and, increasingly, cellulose-active lytic polysaccharide monooxygenases, to deconstruct the recalcitrant β-D-linked polysaccharide. A major drawback with this process is that the exo-acting cellobiohydrolases suffer from severe inhibition from their cellobiose product. β-D-Glucosidases are therefore important for liberating glucose from cellobiose and thereby relieving limiting product inhibition. Here, the three-dimensional structures of two industrially important family GH3 β-D-glucosidases from Aspergillus fumigatus and A. oryzae, solved by molecular replacement and refined at 1.95 Å resolution, are reported. Both enzymes, which share 78% sequence identity, display a three-domain structure with the catalytic domain at the interface, as originally shown for barley β-D-glucan exohydrolase, the first three-dimensional structure solved from glycoside hydrolase family GH3. Both enzymes show extensive N-glycosylation, with only a few external sites being truncated to a single GlcNAc molecule. Those glycans N-linked to the core of the structure are identified purely as high-mannose trees, and establish multiple hydrogen bonds between their sugar components and adjacent protein side chains. The extensive glycans pose special problems for crystallographic refinement, and new techniques and protocols were developed especially for this work. These protocols ensured that all of the D-pyranosides in the glycosylation trees were modelled in the preferred minimum-energy (4)C1 chair conformation and should be of general application to refinements of other crystal structures containing O- or N-glycosylation. The Aspergillus GH3 structures, in light of other recent three-dimensional structures, provide insight into fungal β-D-glucosidases and provide a platform on which to inform and inspire new generations of variant enzymes for industrial application.
authors.affiliation: York Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, England.
authors.forename: Jon
authors.initials: J
authors.lastname: Agirre
authors.affiliation: York Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, England.
authors.forename: Antonio
authors.initials: A
authors.lastname: Ariza
authors.affiliation: York Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, England.
authors.forename: Wendy A
authors.initials: WA
authors.lastname: Offen
authors.affiliation: York Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, England.
authors.forename: Johan P
authors.initials: JP
authors.lastname: Turkenburg
authors.affiliation: York Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, England.
authors.forename: Shirley M
authors.initials: SM
authors.lastname: Roberts
authors.affiliation: York Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, England.
authors.forename: Stuart
authors.initials: S
authors.lastname: McNicholas
authors.affiliation: Novozymes Inc., 1445 Drew Avenue, Davis, CA 95618, USA.
authors.forename: Paul V
authors.initials: PV
authors.lastname: Harris
authors.affiliation: Novozymes Inc., 1445 Drew Avenue, Davis, CA 95618, USA.
authors.forename: Brett
authors.initials: B
authors.lastname: McBrayer
authors.affiliation: York Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, England.
authors.forename: Jan
authors.initials: J
authors.lastname: Dohnalek
authors.affiliation: York Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, England.
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Cowtan
authors.affiliation: York Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, England.
authors.forename: Gideon J
authors.initials: GJ
authors.lastname: Davies
authors.affiliation: York Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, England.
authors.forename: Keith S
authors.initials: KS
authors.lastname: Wilson
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D020134:Catalytic Domain; D002482:Cellulose / Q000737:chemistry; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry*
chemical_list: D005656:Fungal Proteins; D006023:Glycoproteins; D002482:Cellulose; D001617:beta-Glucosidase
keywords: N-glycan; biofuels; cellulose degradation; glucosidase; glycoblocks