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Literature summary for 3.2.1.151 extracted from
- Functional and structural characterization of a potent GH74 endo-xyloglucanase from the soil saprophyte Cellvibrio japonicus unravels the first step of xyloglucan degradation (2016), FEBS J., 283, 1701-1719 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| 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 | Cellvibrio japonicus |
Crystallization (Commentary)
| Crystallization (Comment) | Organism |
|---|---|
| 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 | Cellvibrio japonicus |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| 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 | Cellvibrio japonicus |
| D70A | site-directed mutagenesis, the mutation causes loss of the enzymatic activity by more than 10 000fold compared to the wild-type enzyme | Cellvibrio japonicus |
| 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 | Cellvibrio japonicus |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 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 | Cellvibrio japonicus |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| barley-beta-glucan + H2O | Cellvibrio japonicus | approximately 50fold lower specific activity for the natural mixed-linkage (1->3)/(1->4)-beta-glucan from barley compared to xyloglucan | ? | - |
? |  |
| barley-beta-glucan + H2O | Cellvibrio japonicus Ueda107 | approximately 50fold lower specific activity for the natural mixed-linkage (1->3)/(1->4)-beta-glucan from barley compared to xyloglucan | ? | - |
? | |
| xyloglucan + H2O | Cellvibrio japonicus | 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 | xyloglucooligosaccharides | - |
? | |
| xyloglucan + H2O | Cellvibrio japonicus Ueda107 | 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 | xyloglucooligosaccharides | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Cellvibrio japonicus | B3PKK9 | i.e. Pseudomonas fluorescens subsp. cellulosa | - |
| Cellvibrio japonicus Ueda107 | B3PKK9 | i.e. Pseudomonas fluorescens subsp. cellulosa | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| 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 | Cellvibrio japonicus |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 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 | Cellvibrio japonicus | ? | - |
? | |
| 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 | Cellvibrio japonicus Ueda107 | ? | - |
? | |
| carboxymethyl cellulose + H2O | enzyme CjGH74 shows approximately 165fold lower specific activity for the artificial polysaccharide derivative hydroxyethyl cellulose compared to xyloglucan | Cellvibrio japonicus | ? | - |
? | |
| carboxymethyl cellulose + H2O | enzyme CjGH74 shows approximately 165fold lower specific activity for the artificial polysaccharide derivative hydroxyethyl cellulose compared to xyloglucan | Cellvibrio japonicus Ueda107 | ? | - |
? | |
| hydroxyethyl cellulose + H2O | enzyme CjGH74 shows approximately 24fold lower specific activity for the artificial polysaccharide derivative hydroxyethyl cellulose compared to xyloglucan | Cellvibrio japonicus | ? | - |
? | |
| hydroxyethyl cellulose + H2O | enzyme CjGH74 shows approximately 24fold lower specific activity for the artificial polysaccharide derivative hydroxyethyl cellulose compared to xyloglucan | Cellvibrio japonicus Ueda107 | ? | - |
? | |
| additional information | 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 | Cellvibrio japonicus | ? | - |
? | |
| additional information | 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 | Cellvibrio japonicus Ueda107 | ? | - |
? | |
| tamarind seed xyloglucan + H2O | 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 | Cellvibrio japonicus | xyloglucooligosaccharides | - |
? | |
| xyloglucan + H2O | 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 | Cellvibrio japonicus | xyloglucooligosaccharides | - |
? | |
| xyloglucan + H2O | the endo-xyloglucanase cleaves beta(1->4)-D-glucosidic linkages in the XyG backbone, high specific activity | Cellvibrio japonicus | xyloglucooligosaccharides | - |
? | |
| xyloglucan + H2O | 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 | Cellvibrio japonicus Ueda107 | xyloglucooligosaccharides | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| ? | x * 80811, catalytic module, sequence calculation, x * 80812, catalytic module, mass spectrometry | Cellvibrio japonicus |
| More | 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 | Cellvibrio japonicus |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| CJA_2477 | locus name | Cellvibrio japonicus |
| CjGH74 | - |
Cellvibrio japonicus |
| endo-1,4-beta-glucanase/xyloglucanase | UniProt | Cellvibrio japonicus |
| GH74 endo-xyloglucanase | - |
Cellvibrio japonicus |
| Gly74A | - |
Cellvibrio japonicus |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 65 | 70 | - |
Cellvibrio japonicus |
Temperature Range [°C]
| Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 30 | 90 | activity range, profile overview | Cellvibrio japonicus |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 4.6 | - |
hydroxyethyl cellulose | pH 6.5, 50°C, recombinant enzyme | Cellvibrio japonicus | |
| 77.6 | - |
xyloglucan | pH 6.5, 50°C, recombinant enzyme | Cellvibrio japonicus | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 6 | - |
- |
Cellvibrio japonicus |
pH Range
| pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|
| 3.5 | 8 | activity range, profile overview | Cellvibrio japonicus |
General Information
| General Information | Comment | Organism |
|---|---|---|
| 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) | Cellvibrio japonicus |
| 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 | Cellvibrio japonicus |
| 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 | Cellvibrio japonicus |
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