Literature summary for 3.2.1.139 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.

Search for more literature for 3.2.1.139

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3)	Bacteroides ovatus

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

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

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.3	-	birchwood xylan	recombinant wild-type enzyme, pH 6.5, 37°C, GH30 glucuronoxylan-specific xylanase treated substrate	Bacteroides ovatus
0.4	-	beechwood xylan	recombinant wild-type enzyme, pH 6.5, 37°C	Bacteroides ovatus
0.9	-	beechwood xylan	recombinant wild-type enzyme, pH 6.5, 37°C	Bacteroides ovatus
1.1	-	birchwood xylan	recombinant wild-type enzyme, pH 6.5, 37°C, GH30 glucuronoxylan-specific xylanase treated substrate	Bacteroides ovatus

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
85000	-	2 * 85000, about, sequence calculation	Bacteroides ovatus
199000	-	analytical ultracentrifugation, recombinant wild-type enzyme	Bacteroides ovatus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
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

Organism	UniProt	Comment	Textmining
Bacteroides ovatus	A7M022	-	-

Purification (Commentary)

Purification (Comment)	Organism
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)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
beechwood xylan + H2O	-	Bacteroides ovatus	?	-	?
birchwood xylan + H2O	-	Bacteroides ovatus	?	-	?
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	?	-	?
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

Subunits	Comment	Organism
homodimer	2 * 85000, about, sequence calculation	Bacteroides ovatus
More	the enzyme consists of four distinct domains, which are connected by extended loops, structure overview	Bacteroides ovatus

Synonyms

Synonyms	Comment	Organism
BoAgu115A	-	Bacteroides ovatus
GH115 glucuronidase	-	Bacteroides ovatus

Temperature Optimum [°C]

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

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
11.08	-	birchwood xylan	recombinant wild-type enzyme, pH 6.5, 37°C, GH30 glucuronoxylan-specific xylanase treated substrate	Bacteroides ovatus
11.6	-	beechwood xylan	recombinant wild-type enzyme, pH 6.5, 37°C	Bacteroides ovatus
13.95	-	beechwood xylan	recombinant wild-type enzyme, pH 6.5, 37°C	Bacteroides ovatus
14.37	-	birchwood xylan	recombinant wild-type enzyme, pH 6.5, 37°C, GH30 glucuronoxylan-specific xylanase treated substrate	Bacteroides ovatus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	-	Bacteroides ovatus

General Information

General Information	Comment	Organism
evolution	the enzyme is a member of the glycoside hydrolase family 115, GH115	Bacteroides ovatus
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]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
13.06	-	birchwood xylan	recombinant wild-type enzyme, pH 6.5, 37°C, GH30 glucuronoxylan-specific xylanase treated substrate	Bacteroides ovatus
15.5	-	beechwood xylan	recombinant wild-type enzyme, pH 6.5, 37°C	Bacteroides ovatus
36.95	-	birchwood xylan	recombinant wild-type enzyme, pH 6.5, 37°C, GH30 glucuronoxylan-specific xylanase treated substrate	Bacteroides ovatus
119	-	beechwood xylan	recombinant wild-type enzyme, pH 6.5, 37°C	Bacteroides ovatus