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Information on EC 3.2.1.151 - xyloglucan-specific endo-beta-1,4-glucanase and Organism(s) Cellvibrio japonicus and UniProt Accession B3PKK9
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3.2.1.151 xyloglucan-specific endo-beta-1,4-glucanaseIUBMB Comments
The enzyme from Aspergillus aculeatus is specific for xyloglucan and does not hydrolyse other cell-wall components. The reaction involves endohydrolysis of 1,4-beta-D-glucosidic linkages in xyloglucan with retention of the beta-configuration of the glycosyl residues.
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Word Map
- 3.2.1.151
- xyloglucans
- tamarind
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microfibrils
- xxxg
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expansins
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wall-modifying
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hemicellulosic
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2.4.1.207
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mixed-linkage
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tropaeolum
- nasturtium
- agriculture
- synthesis
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myceliophthora
- degradation
The taxonomic range for the selected organisms is: Cellvibrio japonicus
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Synonyms
xyloglucanase, xyloglucan endotransglucosylase/hydrolases, endoxyloglucanase, xeg12a, xeg5a, mtxgh74, caxth1, cel74a, xeg74, xcxgha, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
endoxyloglucanase
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oligoxyloglucan hydrolase
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XEG
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XG-ase
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XH
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xyloglucan endo-beta-1,4-glucanase
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xyloglucan endohydrolase
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xyloglucan-specific endo 1,4-beta-glucanase
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xyloglucanase
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xyloglucanendohydrolase
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xyloglycan hydrolase
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of O-glycosyl bond
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PATHWAY SOURCE
PATHWAYS
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SYSTEMATIC NAME
IUBMB Comments
[(1->6)-alpha-D-xylo]-(1->4)-beta-D-glucan glucanohydrolase
The enzyme from Aspergillus aculeatus is specific for xyloglucan and does not hydrolyse other cell-wall components. The reaction involves endohydrolysis of 1,4-beta-D-glucosidic linkages in xyloglucan with retention of the beta-configuration of the glycosyl residues.
CAS REGISTRY NUMBER
COMMENTARY
76901-10-5
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
barley-beta-glucan + H2O
?
approximately 50fold lower specific activity for the natural mixed-linkage (1->3)/(1->4)-beta-glucan from barley compared to xyloglucan
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?
carboxymethyl cellulose + H2O
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enzyme CjGH74 shows approximately 165fold lower specific activity for the artificial polysaccharide derivative hydroxyethyl cellulose compared to xyloglucan
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?
hydroxyethyl cellulose + H2O
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enzyme CjGH74 shows approximately 24fold lower specific activity for the artificial polysaccharide derivative hydroxyethyl cellulose compared to xyloglucan
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?
tamarind seed xyloglucan + H2O
xyloglucooligosaccharides
enzyme CjGH74 acting on tamarind XyG reveals that the catalytic module hydrolyzes the polysaccharide at unbranched backbone glucosyl residues to generate the oligosaccharides XXXG, XLXG, XXLG and XLLG, which differ in their degree of side-chain galactosylation. This is the most common cleavage pattern observed for GH74 endo-xyloglucanases
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?
xyloglucan + H2O
xyloglucooligosaccharides
the endo-xyloglucanase cleaves beta(1->4)-D-glucosidic linkages in the XyG backbone, high specific activity, strong preference for xyloglucan as a natural substrate
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?
xyloglucan + H2O
xyloglucooligosaccharides
the endo-xyloglucanase cleaves beta(1->4)-D-glucosidic linkages in the XyG backbone, high specific activity
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?
additional information
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substrate specificity, overview. The enzyme shows no endo-mannanase activity on guar galactomannan and konjac glucomannan, no endoxylanase activity on beechwood xylan and wheat flour arabinoxylan, and no endo-xanthanase on xanthan gum. The recombinant catalytic module indeed demonstrates a strong preference for XyG as a natural substrate. But CjGH74 is unable to release the chromophoric aglycones 2-chloro-4-nitrophenol (CNP) and resorufin from the artificial substrates, XXXG-beta-CNP and XXXG-beta-resorufin. No hydrolysis of the shorter chromogenic substrates CNP-beta-D-cellobioside (GG-beta-CNP) and CNP-beta-D-cellotrioside (GGG-beta-CNP). CjGH74 has an approximately 250 and 970fold higher specificity for xyloglucan compared to the artificial derivative hydroxyethyl cellulose and the mixed-linkage barley-beta-glucan, respectively
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?
additional information
?
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substrate specificity, overview. The enzyme shows no endo-mannanase activity on guar galactomannan and konjac glucomannan, no endoxylanase activity on beechwood xylan and wheat flour arabinoxylan, and no endo-xanthanase on xanthan gum. The recombinant catalytic module indeed demonstrates a strong preference for XyG as a natural substrate. But CjGH74 is unable to release the chromophoric aglycones 2-chloro-4-nitrophenol (CNP) and resorufin from the artificial substrates, XXXG-beta-CNP and XXXG-beta-resorufin. No hydrolysis of the shorter chromogenic substrates CNP-beta-D-cellobioside (GG-beta-CNP) and CNP-beta-D-cellotrioside (GGG-beta-CNP). CjGH74 has an approximately 250 and 970fold higher specificity for xyloglucan compared to the artificial derivative hydroxyethyl cellulose and the mixed-linkage barley-beta-glucan, respectively
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?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
barley-beta-glucan + H2O
?
approximately 50fold lower specific activity for the natural mixed-linkage (1->3)/(1->4)-beta-glucan from barley compared to xyloglucan
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?
xyloglucan + H2O
xyloglucooligosaccharides
the endo-xyloglucanase cleaves beta(1->4)-D-glucosidic linkages in the XyG backbone, high specific activity, strong preference for xyloglucan as a natural substrate
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?
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
Michaelis-Menten kinetics, Km for xyloglucan is 0.008 mg/ml, Vmax is 0.0531 mmol/min/mg, and for hydroxyethyl cellulose Km is 1.2 mg/ml
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additional information
additional information
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Michaelis-Menten kinetics, Km for xyloglucan is 0.008 mg/ml, Vmax is 0.0531 mmol/min/mg, and for hydroxyethyl cellulose Km is 1.2 mg/ml
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
the enzyme belongs to the glycosyl hydrolase family 74, GH74. 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)
metabolism
the enzyme from the saprophytic Gram-negative bacterium catalyzes the first step of xyloglucan degradation. The GH74 catalytic domain generates Glc4-based xyloglucooligosaccharide (XyGO) substrates for downstream enzymes through an endo-dissociative mode of action
additional information
additional information
the substrate binding site of CjGH74 lies in an open cleft at the intersection of the N- and C-terminal domains. The catalytic residues, Asp70 (catalytic base) and Asp483 (catalytic acid), are located on opposite sides in the middle of this cleft. Three-dimensional structure of enzyme CjGH74 in complex with xyloglucooligosaccharides, overview
additional information
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the substrate binding site of CjGH74 lies in an open cleft at the intersection of the N- and C-terminal domains. The catalytic residues, Asp70 (catalytic base) and Asp483 (catalytic acid), are located on opposite sides in the middle of this cleft. Three-dimensional structure of enzyme CjGH74 in complex with xyloglucooligosaccharides, overview
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
x * 80811, catalytic module, sequence calculation, x * 80812, catalytic module, mass spectrometry
additional information
additional information
modular architecture of the native enzyme: the full-length gene product is composed of a signal peptide, a GH74 catalytic domain and two carbohydrate binding modules: CBM10 and CBM2. The GH74, CBM10 and CBM2 modules are connected by serine rich linkers. Calculated molecular masses of the recombinant domains CjGH74-CBM10-CBM2, CjGH74-CBM10, CjGH74, CjCBM10-sfGFP, sfGFP-CjCBM2 and sfGFP are 105.9, 91.2, 80.8, 35.6, 40.7, and 27.8 kDa, respectively. The enzyme structure consists of two seven-bladed beta-propeller domains. Structure comparisons
additional information
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modular architecture of the native enzyme: the full-length gene product is composed of a signal peptide, a GH74 catalytic domain and two carbohydrate binding modules: CBM10 and CBM2. The GH74, CBM10 and CBM2 modules are connected by serine rich linkers. Calculated molecular masses of the recombinant domains CjGH74-CBM10-CBM2, CjGH74-CBM10, CjGH74, CjCBM10-sfGFP, sfGFP-CjCBM2 and sfGFP are 105.9, 91.2, 80.8, 35.6, 40.7, and 27.8 kDa, respectively. The enzyme structure consists of two seven-bladed beta-propeller domains. Structure comparisons
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
apo-CjGH74 comprising residues Pro35 to Ala765 and CjGH74 in complex with Glc4-based xyloglucooligosaccharides, as well as mutant enzymes D70A and D483A, vapour diffusion sitting drop method, from 0.1 M sodium acetate, pH 5.0, and 0.6 M sodium formate, 8% w/v PGA-LM with microseeding, combination of equal volumes of 5 mg/ml protein solution and mother liquor, 19°C, X-ray diffraction structure determination and analysis at 1.71-2.28 A resolution
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D483A
site-directed mutagenesis, a catalytic acid mutant, the mutation causes loss of the enzymatic activity by more than 10 000fold compared to the wild-type enzyme
D70A
site-directed mutagenesis, the mutation causes loss of the enzymatic activity by more than 10 000fold compared to the wild-type enzyme
additional information
additional information
the full-length CJA_2477 gene product is composed of a signal peptide, a GH74 catalytic domain and two carbohydrate binding modules: CBM10 and CBM2. The GH74, CBM10 and CBM2 modules are connected by serine rich linkers. Construction of truncated enzyme forms and isolated domains and recombinant expression as His- and GFP-tagged proteins in Escherichia coli
additional information
-
the full-length CJA_2477 gene product is composed of a signal peptide, a GH74 catalytic domain and two carbohydrate binding modules: CBM10 and CBM2. The GH74, CBM10 and CBM2 modules are connected by serine rich linkers. Construction of truncated enzyme forms and isolated domains and recombinant expression as His- and GFP-tagged proteins in Escherichia coli
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged wild-type enzyme, with a fusion of sfGFP to the C-terminus of carbohydrate binding module CBM10, and enzyme mutants from Escherichia coli strain Rosetta DE3 by nickel affinity chromatography
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
locus CJA_2477 or gene gly74A, DNA and amino acid sequence determination and analysis, recombinant expression of His-tagged enzyme, with a fusion of sfGFP to the C-terminus of carbohydrate binding module CBM10, and enzyme mutants in Escherichia coli strain Rosetta DE3
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Attia, M.; Stepper, J.; Davies, G.J.; Brumer, H.
Functional and structural characterization of a potent GH74 endo-xyloglucanase from the soil saprophyte Cellvibrio japonicus unravels the first step of xyloglucan degradation
FEBS J.
283
1701-1719
2016
Cellvibrio japonicus (B3PKK9), Cellvibrio japonicus, Cellvibrio japonicus Ueda107 (B3PKK9)
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