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

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dc.contributor.authorAttia, Mohamed A.
dc.contributor.authorNelson, Cassandra E.
dc.contributor.authorOffen, Wendy A.
dc.contributor.authorJain, Namrata
dc.contributor.authorDavies, Gideon J.
dc.contributor.authorGardner, Jeffrey G.
dc.contributor.authorBrumer, Harry
dc.date.accessioned2018-03-15T11:58:52Z
dc.date.available2018-03-15T11:58:52Z
dc.date.issued2018
dc.description.abstractBackground 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.en_US
dc.description.urihttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816542/en_US
dc.format.extent16 pagesen_US
dc.genrejournal articlesen_US
dc.identifierdoi:10.13016/M27P8TF9V
dc.identifier.citationAttia MA, Nelson CE, Offen WA, et al. In vitro and in vivo characterization of three Cellvibrio japonicus glycoside hydrolase family 5 members reveals potent xyloglucan backbone-cleaving functions. Biotechnology for Biofuels. 2018;11:45. doi:10.1186/s13068-018-1039-6.en_US
dc.identifier.urihttp://hdl.handle.net/11603/7868
dc.language.isoen_USen_US
dc.publisherBioMed Centralen_US
dc.relation.isAvailableAtThe University of Maryland, Baltimore County (UMBC)
dc.relation.ispartofUMBC Biological Sciences Department Collection
dc.rightsThis item may be protected under Title 17 of the U.S. Copyright Law. It is made available by UMBC for non-commercial research and education. For permission to publish or reproduce, please contact the author.
dc.subjectXyloglucanen_US
dc.subjectSaccharificationen_US
dc.subjectGlycoside hydrolaseen_US
dc.subjectCellvibrio japonicusen_US
dc.subjectSaprophyteen_US
dc.titleIn vitro and in vivo characterization of three Cellvibrio japonicus glycoside hydrolase family 5 members reveals potent xyloglucan backbone-cleaving functionsen_US
dc.typeTexten_US

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