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Literature summary extracted from

  • Rogowski, A.; Basle, A.; Farinas, C.S.; Solovyova, A.; Mortimer, J.C.; Dupree, P.; Gilbert, H.J.; Bolam, D.N.
    Evidence that GH115 alpha-glucuronidase activity, which is required to degrade plant biomass, is dependent on conformational flexibility (2014), J. Biol. Chem., 289, 53-64.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.2.1.131 expressed in Escherichia coli BL21(DE3) cells Bacteroides ovatus
3.2.1.139 recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3) Bacteroides ovatus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.2.1.131 using 19% (w/v) PEG3350, 0.2 M sodium citrate, pH 5.5 Bacteroides ovatus
3.2.1.139 purified recombinant His6-tagged wild-type and selenomethionine-labeled enzyme or enzyme complexed with alpha-D-glucuronic acid, 10 mg/ml protein mixed with 19% PEG3350, 0.2 M sodium citrate, pH 5.5, soaking with 300 mM glucuronic acid for the complexed structure, use of mother liquor supplemented with 15% v/v PEG 400 or paratone N oil as cryoprotectant, X-ray diffraction structure determination and analysis at 2.14-3.0 A resolution Bacteroides ovatus

Protein Variants

EC Number Protein Variants Comment Organism
3.2.1.131 D192A the mutant shows wild type activity Bacteroides ovatus
3.2.1.131 D206A the mutant shows strongly reduced activity compared to the wild type enzyme Bacteroides ovatus
3.2.1.131 D332A inactive Bacteroides ovatus
3.2.1.131 D396N the mutant shows strongly reduced activity compared to the wild type enzyme Bacteroides ovatus
3.2.1.131 D478A the mutant shows wild type activity Bacteroides ovatus
3.2.1.131 E162A the mutant shows wild type activity Bacteroides ovatus
3.2.1.131 E375A almost inactive Bacteroides ovatus
3.2.1.131 E782A the mutant shows reduced activity compared to the wild type enzyme Bacteroides ovatus
3.2.1.131 E785A the mutant shows reduced activity compared to the wild type enzyme Bacteroides ovatus
3.2.1.131 H275A the mutant shows severely reduced activity compared to the wild type enzyme Bacteroides ovatus
3.2.1.131 H275A/H422A inactive Bacteroides ovatus
3.2.1.131 H422A almost inactive Bacteroides ovatus
3.2.1.131 K374A the mutant shows strongly reduced activity compared to the wild type enzyme Bacteroides ovatus
3.2.1.131 N205A the mutant shows strongly reduced activity compared to the wild type enzyme Bacteroides ovatus
3.2.1.131 N398A the mutant shows strongly reduced activity compared to the wild type enzyme Bacteroides ovatus
3.2.1.131 N462A the mutant shows strongly reduced activity compared to the wild type enzyme Bacteroides ovatus
3.2.1.131 R328A almost inactive Bacteroides ovatus
3.2.1.131 W169A the mutant shows wild type activity Bacteroides ovatus
3.2.1.131 W249A the mutant shows severely reduced activity compared to the wild type enzyme Bacteroides ovatus
3.2.1.131 Y373A the mutant shows strongly reduced activity compared to the wild type enzyme Bacteroides ovatus
3.2.1.131 Y420A the mutant shows severely reduced activity compared to the wild type enzyme Bacteroides ovatus
3.2.1.131 Y425A the mutant shows strongly reduced activity compared to the wild type enzyme Bacteroides ovatus
3.2.1.131 Y788A the mutant shows reduced activity compared to the wild type enzyme Bacteroides ovatus
3.2.1.131 Y792A the mutant shows strongly reduced activity compared to the wild type enzyme Bacteroides ovatus
3.2.1.139 D192A site-directed mutagenesis, the mutation has no effect on the enzyme activity Bacteroides ovatus
3.2.1.139 D206A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Bacteroides ovatus
3.2.1.139 D332A site-directed mutagenesis, inactive mutant Bacteroides ovatus
3.2.1.139 D396N site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Bacteroides ovatus
3.2.1.139 D478A site-directed mutagenesis, the mutation has no effect on the enzyme activity Bacteroides ovatus
3.2.1.139 E162A site-directed mutagenesis, the mutation has no effect on the enzyme activity Bacteroides ovatus
3.2.1.139 E375A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Bacteroides ovatus
3.2.1.139 E782A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Bacteroides ovatus
3.2.1.139 E785A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Bacteroides ovatus
3.2.1.139 H275A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Bacteroides ovatus
3.2.1.139 H275A/H422A site-directed mutagenesis, inactive mutant Bacteroides ovatus
3.2.1.139 H422A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Bacteroides ovatus
3.2.1.139 K374A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Bacteroides ovatus
3.2.1.139 additional information deletion of C-terminal residues 1-526, 1-639, and 1-665 results in inactive enzyme mutants Bacteroides ovatus
3.2.1.139 N205A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Bacteroides ovatus
3.2.1.139 N398A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Bacteroides ovatus
3.2.1.139 N462A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Bacteroides ovatus
3.2.1.139 R328A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Bacteroides ovatus
3.2.1.139 W169A site-directed mutagenesis, the mutation has no effect on the enzyme activity Bacteroides ovatus
3.2.1.139 W249A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Bacteroides ovatus
3.2.1.139 Y373A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Bacteroides ovatus
3.2.1.139 Y420A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Bacteroides ovatus
3.2.1.139 Y425A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Bacteroides ovatus
3.2.1.139 Y788A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Bacteroides ovatus
3.2.1.139 Y792A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Bacteroides ovatus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.2.1.131 0.3
-
 birchwood xylan in 50 mM sodium phosphate, 12 mM citrate buffer, pH 6.5, at 37°C Bacteroides ovatus
3.2.1.131 0.4
-
 beechwood xylan in 50 mM sodium phosphate, 12 mM citrate buffer, pH 6.5, at 37°C Bacteroides ovatus
3.2.1.131 4.5
-
 Xyl-beta-(1->4)-[4-O-Me-GlcA-alpha-(1->2)]-Xyl-beta(1->4)-Xyl in 50 mM sodium phosphate, 12 mM citrate buffer, pH 6.5, at 37°C Bacteroides ovatus
3.2.1.131 19.5
-
 4-O-Me-GlcA-alpha-(1->2)-Xyl-beta(1->4)-Xyl in 50 mM sodium phosphate, 12 mM citrate buffer, pH 6.5, at 37°C Bacteroides ovatus
3.2.1.139 0.3
-
 birchwood xylan recombinant wild-type enzyme, pH 6.5, 37°C, GH30 glucuronoxylan-specific xylanase treated substrate Bacteroides ovatus
3.2.1.139 0.4
-
 beechwood xylan recombinant wild-type enzyme, pH 6.5, 37°C Bacteroides ovatus
3.2.1.139 0.9
-
 beechwood xylan recombinant wild-type enzyme, pH 6.5, 37°C Bacteroides ovatus
3.2.1.139 1.1
-
 birchwood xylan recombinant wild-type enzyme, pH 6.5, 37°C, GH30 glucuronoxylan-specific xylanase treated substrate Bacteroides ovatus

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.2.1.131 85000
-
 2 * 85000, calculated from amino acid sequence Bacteroides ovatus
3.2.1.131 199000
-
 analytical ultracentrifugation Bacteroides ovatus
3.2.1.139 85000
-
 2 * 85000, about, sequence calculation Bacteroides ovatus
3.2.1.139 199000
-
 analytical ultracentrifugation, recombinant wild-type enzyme Bacteroides ovatus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.2.1.131 beechwood xylan + H2O Bacteroides ovatus
-
 4-O-methyl-alpha-D-glucuronic acid + D-glucuronic acid + ?
-
 ?
3.2.1.131 beechwood xylan + H2O Bacteroides ovatus GH115
-
 4-O-methyl-alpha-D-glucuronic acid + D-glucuronic acid + ?
-
 ?
3.2.1.131 birchwood xylan + H2O Bacteroides ovatus
-
 4-O-methyl-alpha-D-glucuronic acid + glucuronic acid
-
 ?
3.2.1.131 birchwood xylan + H2O Bacteroides ovatus GH115
-
 4-O-methyl-alpha-D-glucuronic acid + glucuronic acid
-
 ?
3.2.1.139 additional information Bacteroides ovatus the enzyme hydrolyzes methyl-alpha-D-glucuronic acid side chains from the internal regions of xylan. The enzyme is required to undergo a substantial conformational change to form a productive Michaelis complex with glucuronoxylan ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.2.1.131 Bacteroides ovatus
-
-
-
3.2.1.131 Bacteroides ovatus GH115
-
-
-
3.2.1.139 Bacteroides ovatus A7M022
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.2.1.131 immobilized metal affinity chromatography and Superdex 75 gel filtration Bacteroides ovatus
3.2.1.139 recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) to homogeneity by immobilized metal ion affinity chromatography and gel filtration Bacteroides ovatus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.2.1.131 4-O-Me-GlcA-alpha-(1->2)-Xyl-beta(1->4)-Xyl + H2O
-
 Bacteroides ovatus ?
-
 ?
3.2.1.131 4-O-Me-GlcA-alpha-(1->2)-Xyl-beta(1->4)-Xyl + H2O
-
 Bacteroides ovatus GH115 ?
-
 ?
3.2.1.131 beechwood xylan + H2O
-
 Bacteroides ovatus 4-O-methyl-alpha-D-glucuronic acid + D-glucuronic acid + ?
-
 ?
3.2.1.131 beechwood xylan + H2O
-
 Bacteroides ovatus GH115 4-O-methyl-alpha-D-glucuronic acid + D-glucuronic acid + ?
-
 ?
3.2.1.131 birchwood xylan + H2O
-
 Bacteroides ovatus 4-O-methyl-alpha-D-glucuronic acid + glucuronic acid
-
 ?
3.2.1.131 birchwood xylan + H2O
-
 Bacteroides ovatus GH115 4-O-methyl-alpha-D-glucuronic acid + glucuronic acid
-
 ?
3.2.1.131 glucuronoxylan + H2O
-
 Bacteroides ovatus 4-O-methyl-alpha-D-glucuronic acid + ?
-
 ?
3.2.1.131 glucuronoxylan + H2O
-
 Bacteroides ovatus GH115 4-O-methyl-alpha-D-glucuronic acid + ?
-
 ?
3.2.1.131 glucuronoxylan alpha-(4-O-methyl)-glucuronide + H2O
-
 Bacteroides ovatus xylotriose + xylotetraose + ?
-
 ?
3.2.1.131 glucuronoxylan alpha-(4-O-methyl)-glucuronide + H2O
-
 Bacteroides ovatus GH115 xylotriose + xylotetraose + ?
-
 ?
3.2.1.131 Xyl-beta-(1->4)-[4-O-Me-GlcA-alpha-(1->2)]-Xyl-beta(1->4)-Xyl + H2O
-
 Bacteroides ovatus ?
-
 ?
3.2.1.139 beechwood xylan + H2O
-
 Bacteroides ovatus ?
-
 ?
3.2.1.139 birchwood xylan + H2O
-
 Bacteroides ovatus ?
-
 ?
3.2.1.139 additional information the enzyme hydrolyzes methyl-alpha-D-glucuronic acid side chains from the internal regions of xylan. The enzyme is required to undergo a substantial conformational change to form a productive Michaelis complex with glucuronoxylan Bacteroides ovatus ?
-
 ?
3.2.1.139 additional information hydrolysis of the glucurono-xylooligosaccharides derived from mature Arabidopsis thaliana wild-type and gux1gux2 stems as well as wild-type willow, barley, sugar cane, and Miscanthus stems, BoAgu115A is an alpha-glucuronidase that targets the uronic acids that decorate xylans Bacteroides ovatus ?
-
 ?

Subunits

EC Number Subunits Comment Organism
3.2.1.131 homodimer 2 * 85000, calculated from amino acid sequence Bacteroides ovatus
3.2.1.139 homodimer 2 * 85000, about, sequence calculation Bacteroides ovatus
3.2.1.139 More the enzyme consists of four distinct domains, which are connected by extended loops, structure overview Bacteroides ovatus

Synonyms

EC Number Synonyms Comment Organism
3.2.1.131 Agu115A
-
 Bacteroides ovatus
3.2.1.131 alpha-glucuronidase
-
 Bacteroides ovatus
3.2.1.139 BoAgu115A
-
 Bacteroides ovatus
3.2.1.139 GH115 glucuronidase
-
 Bacteroides ovatus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.2.1.139 37
-
 assay at Bacteroides ovatus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.2.1.131 11.1
-
 birchwood xylan in 50 mM sodium phosphate, 12 mM citrate buffer, pH 6.5, at 37°C Bacteroides ovatus
3.2.1.131 11.6
-
 beechwood xylan in 50 mM sodium phosphate, 12 mM citrate buffer, pH 6.5, at 37°C Bacteroides ovatus
3.2.1.131 18.4
-
 4-O-Me-GlcA-alpha-(1->2)-Xyl-beta(1->4)-Xyl in 50 mM sodium phosphate, 12 mM citrate buffer, pH 6.5, at 37°C Bacteroides ovatus
3.2.1.131 53.9
-
 Xyl-beta-(1->4)-[4-O-Me-GlcA-alpha-(1->2)]-Xyl-beta(1->4)-Xyl in 50 mM sodium phosphate, 12 mM citrate buffer, pH 6.5, at 37°C Bacteroides ovatus
3.2.1.139 11.08
-
 birchwood xylan recombinant wild-type enzyme, pH 6.5, 37°C, GH30 glucuronoxylan-specific xylanase treated substrate Bacteroides ovatus
3.2.1.139 11.6
-
 beechwood xylan recombinant wild-type enzyme, pH 6.5, 37°C Bacteroides ovatus
3.2.1.139 13.95
-
 beechwood xylan recombinant wild-type enzyme, pH 6.5, 37°C Bacteroides ovatus
3.2.1.139 14.37
-
 birchwood xylan recombinant wild-type enzyme, pH 6.5, 37°C, GH30 glucuronoxylan-specific xylanase treated substrate Bacteroides ovatus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.2.1.131 7
-
-
 Bacteroides ovatus
3.2.1.139 7
-
-
 Bacteroides ovatus

General Information

EC Number General Information Comment Organism
3.2.1.139 evolution the enzyme is a member of the glycoside hydrolase family 115, GH115 Bacteroides ovatus
3.2.1.139 additional information the crystal structure of BoAgu115A reveals a four-domain protein in which the active site, comprising a pocket that abuts a cleft-like structure, is housed in the second domain that adopts a TIM barrel-fold. The third domain, a five-helical bundle, and the C-terminal beta-sandwich domain make inter-chain contacts leading to protein dimerization, topology of the xylan binding cleft of the enzyme. Active site structure, overview. Residue Arg328 may contribute to substrate binding by also interacting with the carboxylate of the glucuronic acid substrate, residue His422 is a component of the catalytic apparatus Bacteroides ovatus

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.2.1.131 1
-
 4-O-Me-GlcA-alpha-(1->2)-Xyl-beta(1->4)-Xyl in 50 mM sodium phosphate, 12 mM citrate buffer, pH 6.5, at 37°C Bacteroides ovatus
3.2.1.131 12
-
 Xyl-beta-(1->4)-[4-O-Me-GlcA-alpha-(1->2)]-Xyl-beta(1->4)-Xyl in 50 mM sodium phosphate, 12 mM citrate buffer, pH 6.5, at 37°C Bacteroides ovatus
3.2.1.131 29
-
 beechwood xylan in 50 mM sodium phosphate, 12 mM citrate buffer, pH 6.5, at 37°C Bacteroides ovatus
3.2.1.131 37
-
 birchwood xylan in 50 mM sodium phosphate, 12 mM citrate buffer, pH 6.5, at 37°C Bacteroides ovatus
3.2.1.139 13.06
-
 birchwood xylan recombinant wild-type enzyme, pH 6.5, 37°C, GH30 glucuronoxylan-specific xylanase treated substrate Bacteroides ovatus
3.2.1.139 15.5
-
 beechwood xylan recombinant wild-type enzyme, pH 6.5, 37°C Bacteroides ovatus
3.2.1.139 36.95
-
 birchwood xylan recombinant wild-type enzyme, pH 6.5, 37°C, GH30 glucuronoxylan-specific xylanase treated substrate Bacteroides ovatus
3.2.1.139 119
-
 beechwood xylan recombinant wild-type enzyme, pH 6.5, 37°C Bacteroides ovatus