A complex gene locus enables xyloglucan utilization in the model saprophyte <scp><i>C</i></scp><i>ellvibrio japonicus</i>

Larsbrink, Johan; Thompson, Andrew J.; Lundqvist, Magnus; Gardner, Jeffrey G.; Davies, G.J.; Brumer, Harry

doi:10.1111/mmi.12776

Cited by 71 publications

(91 citation statements)

References 75 publications

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“…japonicus represents a useful model for understanding the saprophytic degradation of xyloglucan, one of the most abundant plant cell wall glycans in the biosphere and a key reservoir in the global carbon cycle. Our recent identification of a multigene locus encoding periplasm-localized exo-glycosidases, namely an a-xylosidase, a b-galactosidase and an a-Lfucosidase, in addition to a TBDT provided only a partial picture of XyG utilization by C. japonicus [11]. Here, functional and structural characterization of the multimodular CjGH74-CBM10-CBM2 gene product of locus CJA_2477 sheds new light on the requisite (magenta) (PDB code: 3NDY).…”

Section: Resultsmentioning

confidence: 99%

“…1) [10]. As a result of this structural complexity, complete XyG saccharification requires a consortium of endo-acting and exoacting glycoside hydrolases [11,12].…”

Section: Introductionmentioning

confidence: 99%

“…We recently identified and characterized a multicomponent gene locus in C. japonicus that is uniquely responsible for XyG utilization (XyGUL) [11,22]. This locus (CJA2706-2710) encodes all of the exo-glycosidases required for the cleavage of the branches of dicot (fucogalacto)xyloglucan, namely a glycoside hydrolase family 95 (GH95) a-L-fucosidase, a GH35 bgalactosidase and a GH31 a-xylosidase, as well as a predicted TonB-dependent transporter (TBDT) [11,22,23].…”

Section: Introductionmentioning

confidence: 99%

“…This locus (CJA2706-2710) encodes all of the exo-glycosidases required for the cleavage of the branches of dicot (fucogalacto)xyloglucan, namely a glycoside hydrolase family 95 (GH95) a-L-fucosidase, a GH35 bgalactosidase and a GH31 a-xylosidase, as well as a predicted TonB-dependent transporter (TBDT) [11,22,23]. However, this locus does not encode an endo-xyloglucanase, which is required for the initial generation of xylogluco-oligosaccharides (XyGOs) as substrates for these exo-glycosidases.…”

Section: Introductionmentioning

confidence: 99%

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Functional and structural characterization of a potent GH74 endo‐xyloglucanase from the soil saprophyte Cellvibrio japonicus unravels the first step of xyloglucan degradation

et al. 2016

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The heteropolysaccharide xyloglucan (XyG) comprises up to one‐quarter of the total carbohydrate content of terrestrial plant cell walls and, as such, represents a significant reservoir in the global carbon cycle. The complex composition of XyG requires a consortium of backbone‐cleaving endo‐xyloglucanases and side‐chain cleaving exo‐glycosidases for complete saccharification. The biochemical basis for XyG utilization by the model Gram‐negative soil saprophytic bacterium Cellvibrio japonicus is incompletely understood, despite the recent characterization of associated side‐chain cleaving exo‐glycosidases. We present a detailed functional and structural characterization of a multimodular enzyme encoded by gene locus CJA_2477. The CJA_2477 gene product comprises an N‐terminal glycoside hydrolase family 74 (GH74) endo‐xyloglucanase module in train with two carbohydrate‐binding modules (CBMs) from families 10 and 2 (CBM10 and CBM2). The GH74 catalytic domain generates Glc4‐based xylogluco‐oligosaccharide (XyGO) substrates for downstream enzymes through an endo‐dissociative mode of action. X‐ray crystallography of the GH74 module, alone and in complex with XyGO products spanning the entire active site, revealed a broad substrate‐binding cleft specifically adapted to XyG recognition, which is composed of two seven‐bladed propeller domains characteristic of the GH74 family. The appended CBM10 and CBM2 members notably did not bind XyG, nor other soluble polysaccharides, and instead were specific cellulose‐binding modules. Taken together, these data shed light on the first step of xyloglucan utilization by C. japonicus and expand the repertoire of GHs and CBMs for selective biomass analysis and utilization. Database Structural data have been deposited in the RCSB protein database under the Protein Data Bank codes: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5FKR, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5FKS, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5FKT and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5FKQ.

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Section: Resultsmentioning

confidence: 99%

“…1) [10]. As a result of this structural complexity, complete XyG saccharification requires a consortium of endo-acting and exoacting glycoside hydrolases [11,12].…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Functional and structural characterization of a potent GH74 endo‐xyloglucanase from the soil saprophyte Cellvibrio japonicus unravels the first step of xyloglucan degradation

et al. 2016

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“…XXXG was prepared similarly, with CjBgl35A replacing the A. niger ␤-galactosidase (33). Briefly, 100 g of dTKP were slowly added to 1 liter of 10 mM NH 4 OAc (pH 5.5) containing 500 units (ϳ2.5 mg) of PpXG5 (where 1 unit is defined as the amount of enzyme that releases 1 mol of glucose-eq reducing ends per min).…”

Section: Substrates and Inhibitorsmentioning

confidence: 99%

Structure-Function Analysis of a Mixed-linkage β-Glucanase/Xyloglucanase from the Key Ruminal Bacteroidetes Prevotella bryantii B14

McGregor

Morar

Fenger

et al. 2016

Journal of Biological Chemistry

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The recent classification of glycoside hydrolase family 5 (GH5) members into subfamilies enhances the prediction of substrate specificity by phylogenetic analysis. However, the small number of well characterized members is a current limitation to understanding the molecular basis of the diverse specificity observed across individual GH5 subfamilies. GH5 subfamily 4 (GH5_4) is one of the largest, with known activities comprising (carboxymethyl)cellulases, mixedlinkage endo-glucanases, and endo-xyloglucanases. Through detailed structure-function analysis, we have revisited the characterization of a classic GH5_4 carboxymethylcellulase, PbGH5A (also known as Orf4, carboxymethylcellulase, and Cel5A), from the symbiotic rumen Bacteroidetes Prevotella bryantii B 1 4. We demonstrate that carboxymethylcellulose and phosphoric acid-swollen cellulose are in fact relatively poor substrates for PbGH5A, which instead exhibits clear primary specificity for the plant storage and cell wall polysaccharide, mixedlinkage ␤-glucan. Significant activity toward the plant cell wall polysaccharide xyloglucan was also observed. Determination of PbGH5A crystal structures in the apo-form and in complex with (xylo)glucan oligosaccharides and an active-site affinity label, together with detailed kinetic analysis using a variety of well defined oligosaccharide substrates, revealed the structural determinants of polysaccharide substrate specificity. In particular, this analysis highlighted the PbGH5A active-site motifs that engender predominant mixed-linkage endo-glucanase activity vis à vis predominant endo-xyloglucanases in GH5_4. However the detailed phylogenetic analysis of GH5_4 members did not delineate particular clades of enzymes sharing these sequence motifs; the phylogeny was instead dominated by bacterial taxonomy. Nonetheless, our results provide key enzyme functional and structural reference data for future bioinformatics analyses of (meta)genomes to elucidate the biology of complex gut ecosystems.

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Characterization of an α‐L‐fucosidase in marine bacterium Wenyingzhuangia fucanilytica: new evidence on the catalytic sites of GH95 family glycosidases

Shen,

Li,

Zhang

et al. 2024

J Sci Food Agric

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Backgroundα‐l‐Fucose confers unique functions for fucose‐containing biomolecules such as human milk oligosaccharides. α‐l‐Fucosidases can serve as desirable tools in the application of fucosylated saccharides. Discovering novel α‐l‐fucosidases and elucidating their enzyme properties are always worthy tasks.ResultsA GH95 family α‐l‐fucosidase named Afc95A_Wf was cloned from the genome of the marine bacterium Wenyingzhuangia fucanilytica and expressed in Escherichia coli. It exhibited maximum activity at 40 °C and pH 7.5. Afc95A_Wf defined a different substrate specificity among reported α‐l‐fucosidases, which was capable of hydrolyzing α‐fucoside in CNP‐fucose, Fucα1‐2Galβ1‐4Glc and Galβ1‐4(Fucα1‐3)Glc, and showed a preference for α1,2‐fucosidic linkage. It adopted Asp residue in the amino acid sequence at position 391, which was distinct from the previously acknowledged residue of Asn. The predicted tertiary structure and site‐directed mutagenesis revealed that Asp391 participates in the catalysis of Afc95A_Wf. The differences in the substrate specificity and catalytic site shed light on that Afc95A_Wf adopted a novel mechanism in catalysis.ConclusionA GH95 family α‐l‐fucosidase (Afc95A_Wf) was cloned and expressed. It showed a cleavage preference for α1,2‐fucosidic linkage to α1,3‐fucosidic linkage. Afc95A_Wf demonstrated a different substrate specificity and a residue at an important catalytic site compared with known GH95 family proteins, which revealed the occurrence of diversity on catalytic mechanisms in the GH95 family. © 2024 Society of Chemical Industry.

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A complex gene locus enables xyloglucan utilization in the model saprophyte Cellvibrio japonicus

Cited by 71 publications

References 75 publications

Functional and structural characterization of a potent GH74 endo‐xyloglucanase from the soil saprophyte Cellvibrio japonicus unravels the first step of xyloglucan degradation

Functional and structural characterization of a potent GH74 endo‐xyloglucanase from the soil saprophyte Cellvibrio japonicus unravels the first step of xyloglucan degradation

Structure-Function Analysis of a Mixed-linkage β-Glucanase/Xyloglucanase from the Key Ruminal Bacteroidetes Prevotella bryantii B14

Characterization of an α‐L‐fucosidase in marine bacterium Wenyingzhuangia fucanilytica: new evidence on the catalytic sites of GH95 family glycosidases

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