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In vitro and in vivo characterization of three Cellvibrio japonicus glycoside hydrolase family 5 members reveals potent xyloglucan backbone-cleaving functions

DOI: 10.1186/s13068-018-1039-6 DOI Help

Authors: Mohamed A. Attia (Michael Smith Laboratories, University of British Columbia) , Cassandra E. Nelson (University of Maryland) , Wendy A. Offen (University of York) , Namrata Jain (Michael Smith Laboratories, University of British Columbia) , Gideon J. Davies (University of York) , Jeffrey G. Gardner (University of Maryland) , Harry Brumer (Michael Smith Laboratories, University of British Columbia)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biotechnology For Biofuels , VOL 11

State: Published (Approved)
Published: February 2018
Diamond Proposal Number(s): 9948

Open Access Open Access

Abstract: Background: Xyloglucan (XyG) is a ubiquitous and fundamental polysaccharide of plant cell walls. Due to its structural complexity, XyG requires a combination of backbone-cleaving and sidechain-debranching enzymes for complete deconstruction into its component monosaccharides. The soil saprophyte Cellvibrio japonicus has emerged as a genetically tractable model system to study biomass saccharification, in part due to its innate capacity to utilize a wide range of plant polysaccharides for growth. Whereas the downstream debranching enzymes of the xyloglucan utilization system of C. japonicus have been functionally characterized, the requisite backbone-cleaving endo-xyloglucanases were unresolved. Results: Combined bioinformatic and transcriptomic analyses implicated three glycoside hydrolase family 5 subfamily 4 (GH5_4) members, with distinct modular organization, as potential keystone endo-xyloglucanases in C. japonicus. Detailed biochemical and enzymatic characterization of the GH5_4 modules of all three recombinant proteins confirmed particularly high specificities for the XyG polysaccharide versus a panel of other cell wall glycans, including mixed-linkage beta-glucan and cellulose. Moreover, product analysis demonstrated that all three enzymes generated XyG oligosaccharides required for subsequent saccharification by known exo-glycosidases. Crystallographic analysis of GH5D, which was the only GH5_4 member specifically and highly upregulated during growth on XyG, in free, product-complex, and active-site affinity-labelled forms revealed the molecular basis for the exquisite XyG specificity among these GH5_4 enzymes. Strikingly, exhaustive reverse-genetic analysis of all three GH5_4 members and a previously biochemically characterized GH74 member failed to reveal a growth defect, thereby indicating functional compensation in vivo, both among members of this cohort and by other, yet unidentified, xyloglucanases in C. japonicus. Our systems-based analysis indicates distinct substrate-sensing (GH74, GH5E, GH5F) and attack-mounting (GH5D) functions for the endo-xyloglucanases characterized here. Conclusions: Through a multi-faceted, molecular systems-based approach, this study provides a new insight into the saccharification pathway of xyloglucan utilization system of C. japonicus. The detailed structural–functional characterization of three distinct GH5_4 endo-xyloglucanases will inform future bioinformatic predictions across species, and provides new CAZymes with defined specificity that may be harnessed in industrial and other biotechnological applications.

Journal Keywords: Xyloglucan; Saccharification; Glycoside hydrolase; Cellvibrio japonicus; Saprophyte

Diamond Keywords: Biofuel; Enzymes

Subject Areas: Biology and Bio-materials, Energy, Chemistry

Instruments: I03-Macromolecular Crystallography , I04-Macromolecular Crystallography

Added On: 22/02/2018 10:46


Discipline Tags:

Catalysis Earth Sciences & Environment Climate Change Energy Bioenergy Sustainable Energy Systems Engineering & Technology Biotechnology Life Sciences & Biotech Structural biology Chemistry Biochemistry

Technical Tags:

Diffraction Macromolecular Crystallography (MX)