Three-dimensional structures of two heavily N-glycosylated Aspergillus sp family GH3 beta-D-glucosidases

0470039 - BTÚ 2017 RIV GB eng J - Journal Article
Agirre, J. - Ariza, A. - Offen, W. A. - Turkenburg, J. P. - Roberts, S. M. - McNicholas, S. - Harris, P. V. - McBrayer, B. - Dohnálek, Jan - Cowtan, K. D. - Davies, G. J. - Wilson, K. S.
Three-dimensional structures of two heavily N-glycosylated Aspergillus sp family GH3 beta-D-glucosidases.
Acta Crystallographica Section D-Structural Biology. Roč. 72, č. 2 (2016), s. 254-265. ISSN 2059-7983. E-ISSN 2059-7983
R&D Projects: GA MŠMT(CZ) ED1.1.00/02.0109
Institutional support: RVO:86652036
Keywords : cellulose degradation * N-glycan * biofuels * glycoblocks
Subject RIV: EB - Genetics ; Molecular Biology
Impact factor: 2.114, year: 2016

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 beta-D-linked polysaccharide. A major drawback with this process is that the exo-acting cellobiohydrolases suffer from severe inhibition from their cellobiose product. beta-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 beta-D-glucosidases from Aspergillus fumigatus and A. oryzae, solved by molecular replacement and refined at 1.95 angstrom resolution, are reported. Both enzymes display a three-domain structure with the catalytic domain at the interface, as originally shown for barley beta-D-glucan exohydrolase. 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 C-4(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 beta-D-glucosidases and provide a platform on which to inform and inspire new generations of variant enzymes for industrial application.
Permanent Link: http://hdl.handle.net/11104/0267784