Attia, Mohamed A., Nelson, Cassandra E., Offen, Wendy A. orcid.org/0000-0002-2758-4531 et al. (4 more authors) (2018) In vitro and in vivo characterization of three Cellvibrio japonicus Glycoside Hydrolase Family 5 members reveals potent xyloglucan backbone-cleaving functions. Biotechnology for biofuels. 45. ISSN 1754-6834
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.
Metadata
Item Type: | Article |
---|---|
Authors/Creators: |
|
Copyright, Publisher and Additional Information: | © The Author(s) 2018. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. Further copying may not be permitted; contact the publisher for details |
Keywords: | Cellvibrio japonicus,Glycoside hydrolase,Saccharification,Saprophyte,Xyloglucan |
Dates: |
|
Institution: | The University of York |
Academic Units: | The University of York The University of York > Faculty of Sciences (York) > Chemistry (York) |
Funding Information: | Funder Grant number BBSRC (BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL) BB/I014802/1 |
Depositing User: | Pure (York) |
Date Deposited: | 31 Jan 2018 17:00 |
Last Modified: | 17 Dec 2024 00:09 |
Published Version: | https://doi.org/10.1186/s13068-018-1039-6 |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.1186/s13068-018-1039-6 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:126900 |