Three-dimensional structures of two heavily N-glycosylatedAspergillussp. 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 -d-linked polysaccharide. A major drawback with this process is that the exo-acting cellobiohydrolases suffer from severe inhibition from their cellobiose product. -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 -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 -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 highmannose 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 C 1 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 -d-glucosidases and provide a platform on which to inform and inspire new generations of variant enzymes for industrial application.

Structural and Functional Characterization of a Ruminal β-Glycosidase Defines a Novel Subfamily of Glycoside Hydrolase Family 3 with Permuted Domain Topology

“…2 A ); both of them provided residues that make up the active site. The later reported structures from T. neapolitana (8), Trichoderma reesei (12), Aspergillus (13, 14), and L. innocua (18) β-glucosidases, and a β-glucosidase isolated from soil compost (32), showed the presence of an additional fibronectin type III (FnIII) domain (also designated fibronectin-like domain or FLD) located at the C terminus. This three-domain arrangement is shared by other reported β-glucosidases from K. marxianus (9) and Streptomyces venezuelae (11) that also contain an additional PA14 domain inserted within the same loop of their (α/β) 6 -sandwich, although both are arranged in a different orientation.…”

Carbohydrate structure: the rocky road to automation

“…Strangely, mannose 6 was fitted in a wrong orientation, thus ended up completely distorted and disconnected from the rest. An additional mannose (7) was added, which the depositors had decided not to model due to unclear electron density, and this achieved a low RSCC of 0.64. b. Modelling a high-mannose glycan in a 1.8 •-resolution map (nativelyglycosylated fungal GH3 glycosyl hydrolase [26], PDB code 5FJI). The program was able to trace a glycan that matched the deposited one very closely, although it modelled an additional mannose (5) for which density was scarce.…”

Carbohydrate 3D structure validation

“…Upon detection of high-energy conformations, the software creates dictionaries containing aperiodic torsion restraints in standard CIF format, which can be read by most model building and refinement programs [17]. Privateer has been used successfully to detect and prevent conformational anomalies on medium [26,27] (also shown in Fig. 1) and low resolution [28] crystallographic structures, and more recently on cryoEM data [29].…”

“…COOT) for iterative validation. For completeness, the influence of interactions (H-bond [31], stacking [32]) on the selection of alternative link energy minima [26,33] should also be reflected. Such is the case of the GlcNAc-Asn bond in N-glycosylation (Fig.…”

“…Such is the case of the GlcNAc-Asn bond in N-glycosylation (Fig. 1, bottom panel): a secondary energy minimum can be found at 45¼ < j N < 110¼, which may be selected based on neighbouring interactions [26].…”