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Literature summary extracted from

  • Arnal, G.; Stogios, P.J.; Asohan, J.; Skarina, T.; Savchenko, A.; Brumer, H.
    Structural enzymology reveals the molecular basis of substrate regiospecificity and processivity of an exemplar bacterial glycoside hydrolase family 74 endo-xyloglucanase (2018), Biochem. J., 475, 3963-3978 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.2.1.151 DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression of N-terminally His6-tagged wild-type and mutant catalytic domains in Escherichia coli strain BL21(DE3) Paenibacillus odorifer
3.2.1.151 recombinant expression of His-tagged wild-type and mutant catalytic PoGH74cat enzyme modules in Escherichia coli strain BL21(DE3) Paenibacillus odorifer

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.2.1.151 PoGH74cat as apoenzyme and as XLX complex, sitting drop vapour diffusion method, 0.0015 ml of 20 mg/ml protein with 10 mM xylose and 0.1 M glucose are mixed with 0.0015 ml of the reservoir solution 25% w/v PEG 8000, 100 mM Bis-Tris buffer, pH 6.9, 1 mM tris(2-carboxyethyl)phosphine, and 0.2 M sodium acetate, crystals of PoGH74cat in complex with XLX are grown by cocrystallization using 500 nl of 10 mg/ml protein plus 5 mM of a xyloglucan oligosaccharide mixture (XXXG, XLXG, XXLG, and XLLG) mixed with 500 nl of the reservoir solution 20% w/v PEG3350 and 0.2 M sodium potassium tartrate. Crystals of the PoGH74catD70A mutant in complex with XXLG and XGXXLG are obtained by cocrystallization of 500 nl of 10 mg/ml protein with a complex XyGO mixture mixed with 500 nl reservoir solution 25% w/v PEG3350, 0.2 M magnesium chloride, and 100 mM Tris buffer, pH 8.5, 22°C, X-ray diffraction structure determination and analysis at 1.50-2.10 A resolution, molecular replacement and modeling Paenibacillus odorifer

Protein Variants

EC Number Protein Variants Comment Organism
3.2.1.151 D70A site-directed mutagenesis in the PoGH74cat module at the catalytic base Paenibacillus odorifer
3.2.1.151 D70A site-directed mutagenesis, mutation of the catalytic base, the mutant turnover rate is similar to wild-type Paenibacillus odorifer
3.2.1.151 G476Y site-directed mutagenesis in the PoGH74cat module at the -1 subsite Paenibacillus odorifer
3.2.1.151 G476Y site-directed mutagenesis, mutation of the -1 subsite, the mutant turnover rate is similar to wild-type Paenibacillus odorifer
3.2.1.151 additional information generation of PoGH74cat(DELTA642-651) and PoGH74cat(DELTA671-675) truncated mutants Paenibacillus odorifer
3.2.1.151 additional information construction of truncated mutants, i.e. PoGH74cat(DELTA642-651) and PoGH74cat(DELTA671-675), the mutant turnover rate is similar to wild-type Paenibacillus odorifer
3.2.1.151 W347A site-directed mutagenesis in the PoGH74cat module at the +3 subsite Paenibacillus odorifer
3.2.1.151 W347A site-directed mutagenesis, mutation of the +3 subsite, kcat value is increased by 5fold compared to wild-type Paenibacillus odorifer
3.2.1.151 W348A site-directed mutagenesis in the PoGH74cat module at the +5 subsite Paenibacillus odorifer
3.2.1.151 W348A site-directed mutagenesis, mutation of the +5 subsite, the mutant turnover rate is similar to wild-type Paenibacillus odorifer
3.2.1.151 W406A site-directed mutagenesis in the PoGH74cat module at the +2 subsite Paenibacillus odorifer
3.2.1.151 W406A site-directed mutagenesis, mutation of the +2 subsite, the mutant turnover rate is similar to wild-type Paenibacillus odorifer
3.2.1.151 Y372A site-directed mutagenesis in the PoGH74cat module at the +6 subsite Paenibacillus odorifer
3.2.1.151 Y372A site-directed mutagenesis, mutation of the +6 subsite, the mutant turnover rate is similar to wild-type Paenibacillus odorifer

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.2.1.151 additional information
-
 additional information Michaelis-Menten kinetics Paenibacillus odorifer
3.2.1.151 additional information
-
 additional information Michaelis-Menten kinetics, Km values for wild-type and mutant enzymes are 0.04-0.23 mg/ml Paenibacillus odorifer

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.2.1.151 additional information Paenibacillus odorifer endo-xyloglucanases from GH74 can hydrolyze the regular structure of XXXG-type XyGs at the anomeric center of the unbranched glucosyl (G) unit, although some cleave the backbone at more sterically encumbered positions, e.g. between two X units ?
-
 ?
3.2.1.151 additional information Paenibacillus odorifer DSM 15391 endo-xyloglucanases from GH74 can hydrolyze the regular structure of XXXG-type XyGs at the anomeric center of the unbranched glucosyl (G) unit, although some cleave the backbone at more sterically encumbered positions, e.g. between two X units ?
-
 ?
3.2.1.151 xyloglucan + H2O Paenibacillus odorifer
-
 xyloglucan oligosaccharides
-
 ?
3.2.1.151 xyloglucan + H2O Paenibacillus odorifer DSM 15391
-
 xyloglucan oligosaccharides
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.2.1.151 Paenibacillus odorifer A0A1R0YRH0
-
-
3.2.1.151 Paenibacillus odorifer A0A1R0ZNW0
-
-
3.2.1.151 Paenibacillus odorifer DSM 15391 A0A1R0YRH0
-
-
3.2.1.151 Paenibacillus odorifer DSM 15391 A0A1R0ZNW0
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.2.1.151 recombinant His-tagged wild-type and mutant catalytic PoGH74cat enzyme modules from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, ultrafiltration, dialysis, and gel filtration Paenibacillus odorifer
3.2.1.151 recombinant N-terminally His6-tagged wild-type and mutant catalytic domains from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage by TEV protease, ultrafiltration, and gel filtration Paenibacillus odorifer

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.2.1.151 additional information endo-xyloglucanases from GH74 can hydrolyze the regular structure of XXXG-type XyGs at the anomeric center of the unbranched glucosyl (G) unit, although some cleave the backbone at more sterically encumbered positions, e.g. between two X units Paenibacillus odorifer ?
-
 ?
3.2.1.151 additional information residue Gly476 is uniquely responsible for the promiscuous xyloglucan backbone-cleaving activity of the GH74 module PoGH74cat Paenibacillus odorifer ?
-
 ?
3.2.1.151 additional information enzymatic assays are performed on AZCL-xyloglucan, release of soluble dye fragments from cross-linked AZCL-XyG is measured. Enzyme PoGH74 shows essentially exclusive specificity for xyloglucan (XyG), which is typical for all GH74 members Paenibacillus odorifer ?
-
-
3.2.1.151 additional information enzymatic assays are performed on AZCL-xyloglucan, release of soluble dye fragments from cross-linked AZCL-XyG is measured. Enzyme PoGH74 shows essentially exclusive specificity for xyloglucan (XyG), which is typical for all GH74 members Paenibacillus odorifer DSM 15391 ?
-
-
3.2.1.151 additional information endo-xyloglucanases from GH74 can hydrolyze the regular structure of XXXG-type XyGs at the anomeric center of the unbranched glucosyl (G) unit, although some cleave the backbone at more sterically encumbered positions, e.g. between two X units Paenibacillus odorifer DSM 15391 ?
-
 ?
3.2.1.151 additional information residue Gly476 is uniquely responsible for the promiscuous xyloglucan backbone-cleaving activity of the GH74 module PoGH74cat Paenibacillus odorifer DSM 15391 ?
-
 ?
3.2.1.151 xyloglucan + H2O the GH74 module (PoGH74cat) reveals a highly specific, processive endo-xyloglucanase activity that can hydrolyze the polysaccharide backbone at both branched and unbranched positions Paenibacillus odorifer ?
-
 ?
3.2.1.151 xyloglucan + H2O the GH74 module (PoGH74cat) reveals a highly specific, processive endo-xyloglucanase activity that can hydrolyze the polysaccharide backbone at both branched and unbranched positions Paenibacillus odorifer DSM 15391 ?
-
 ?
3.2.1.151 xyloglucan + H2O
-
 Paenibacillus odorifer xyloglucan oligosaccharides
-
 ?
3.2.1.151 xyloglucan + H2O xyloglucan from tamarind seeds, structural analysis of xyloglucan binding structure with PoGH74cat, and structural basis of the bond cleavage pattern of PoGH74cat, overview Paenibacillus odorifer xyloglucan oligosaccharides
-
 ?
3.2.1.151 xyloglucan + H2O
-
 Paenibacillus odorifer DSM 15391 xyloglucan oligosaccharides
-
 ?
3.2.1.151 xyloglucan + H2O xyloglucan from tamarind seeds, structural analysis of xyloglucan binding structure with PoGH74cat, and structural basis of the bond cleavage pattern of PoGH74cat, overview Paenibacillus odorifer DSM 15391 xyloglucan oligosaccharides
-
 ?

Subunits

EC Number Subunits Comment Organism
3.2.1.151 More mature PoGH74 is composed of an N-terminal GH74 catalytic module in-train with three modules of unknown function (X2 domain, PFAM03442, and a carbohydrate-binding module (CBM) family 3), modular architecture of the native Paenibacillus odorifer AIQ73809 gene product, and GH74 enzymes structure comparisons, overview Paenibacillus odorifer

Synonyms

EC Number Synonyms Comment Organism
3.2.1.151 bacterial glycoside hydrolase family 74 endo-xyloglucanase
-
 Paenibacillus odorifer
3.2.1.151 endo-xyloglucanase
-
 Paenibacillus odorifer
3.2.1.151 PODO_RS11130 locus name Paenibacillus odorifer
3.2.1.151 PoGH74
-
 Paenibacillus odorifer

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.2.1.151 37
-
 assay at Paenibacillus odorifer

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.2.1.151 25.3
-
 xyloglucan xyloglucan from tamarind seed, pH 7.0, 37°C, recombinant mutant PoGH74catG476Y Paenibacillus odorifer
3.2.1.151 39.8
-
 xyloglucan xyloglucan from tamarind seed, pH 7.0, 37°C, recombinant wild-type enzyme PoGH74 Paenibacillus odorifer
3.2.1.151 43.8
-
 xyloglucan xyloglucan from tamarind seed, pH 7.0, 37°C, recombinant mutant PoGH74catW406A Paenibacillus odorifer
3.2.1.151 47.5
-
 xyloglucan xyloglucan from tamarind seed, pH 7.0, 37°C, recombinant mutant PoGH74catY372A Paenibacillus odorifer
3.2.1.151 58.3
-
 xyloglucan xyloglucan from tamarind seed, pH 7.0, 37°C, recombinant mutant PoGH74catW348A Paenibacillus odorifer
3.2.1.151 214.7
-
 xyloglucan xyloglucan from tamarind seed, pH 7.0, 37°C, recombinant mutant PoGH74catW347A Paenibacillus odorifer

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.2.1.151 7
-
 assay at Paenibacillus odorifer

General Information

EC Number General Information Comment Organism
3.2.1.151 evolution Paenibacillus odorifer produces a single multimodular enzyme containing a glycoside hydrolase (GH) family 74 module (AIQ73809). Endo-xyloglucanases, which catalyze the cleavage of the xyloglucans (XyGs) backbone (EC 3.2.1.151), are currently found in glycoside hydrolase (GH) families GH5, GH9, GH12, GH16, GH44, and GH74 (in CAZy classification). Of these, family GH74 is distinguished by fewer sequence members, an essentially singular specificity for xyloglucans, and a characteristic tertiary structure comprised of two 7-bladed beta-propeller domains that form a large interfacial cleft to accommodate the bulky polysaccharide Paenibacillus odorifer
3.2.1.151 evolution the endo-xyloglucanase belongs to the bacterial glycoside hydrolase family 74, GH74. Endo-xyloglucanases, which catalyze the cleavage of the XyGs backbone (EC 3.2.1.151), are currently found in glycoside hydrolase (GH) families GH5, GH9, GH12, GH16, GH44, and GH74 in the carbohydrate-active enzymes (CAZy) classification. Of these, family GH74 is distinguished by fewer sequence members, an essentially singular specificity for XyGs, and a characteristic tertiary structure comprised of two 7-bladed beta-propeller domains that form a large interfacial cleft to accommodate the bulky polysaccharide. Structure-activity relationships among characterized GH74 members, including determinants of endo versus exo (EC 3.2.1.150) activity, have been reviewed Paenibacillus odorifer
3.2.1.151 malfunction replacement of catalytic Gly476 with Tyr, which is conserved in many GH74 members, results in exclusive hydrolysis of xyloglucan at unbranched glucose units. Likewise, systematic replacement of the hydrophobic platform residues constituting the positive subsites indicated their relative contributions to the processive mode of action. Specifically, W347 (+3 subsite) and W348 (+5 subsite) are essential for processivity, while W406 (+2 subsite) and Y372 (+6 subsite) are not strictly essential, but aid processivity Paenibacillus odorifer
3.2.1.151 physiological function endo-xyloglucanases from the GH74 family hydrolyze xyloglucans (XyGs). Enzymatic XyG hydrolysis increases cellulose digestibility, which underscores their structural importance in the plant cell wall. XyGs are also found as storage polysaccharides in some seeds, e.g. tamarind, and therefore represent important agricultural byproducts that have applications in the food, biomaterial and medical sectors. The endo-xyloglucanase can hydrolyze the regular structure of XXXG-type XyGs at the anomeric center of the unbranched glucosyl (G) unit, although some cleave the backbone at more sterically encumbered positions, e.g. between two X units Paenibacillus odorifer