Information on EC 3.2.1.156 - oligosaccharide reducing-end xylanase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.2.1.156
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RECOMMENDED NAME
GeneOntology No.
oligosaccharide reducing-end xylanase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Hydrolysis of (1->4)-beta-D-xylose residues from the reducing end of oligosaccharides
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of O-glycosyl bond
SYSTEMATIC NAME
IUBMB Comments
beta-D-xylopyranosyl-(1->4)-beta-D-xylopyranose reducing-end xylanase
The enzyme, originally isolated from the bacterium Bacillus halodurans C-125, releases the xylose unit at the reducing end of oligosaccharides ending with the structure beta-D-xylopyranosyl-(1->4)-beta-D-xylopyranosyl-(1->4)-beta-D-xylopyranose, leaving the new reducing end in the alpha configuration. It is specific for the beta anomers of xylooligosaccharides whose degree of polymerization is equal to or greater than 3. The penultimate residue must be beta-D-xylopyranose, but replacing either of the flanking residues with glucose merely slows the rate greatly.
CAS REGISTRY NUMBER
COMMENTARY hide
55126-95-9
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879497-03-7
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1-O-fluoro-4-O-alpha-D-xylopyranosyl-beta-D-xylopyranose + H2O
alpha-D-xylopyranose + 1-O-fluoro-beta-D-xylopyranose
show the reaction diagram
2-nitrohenyl Xylbeta(1-4)Xylbeta(1-4)Xylbeta(1-4)Xyl + H2O
2-nitrophenol + Xylbeta(1-4)Xylbeta(1-4)Xylbeta(1-4)Xyl
show the reaction diagram
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-
-
?
2-nitrophenyl Xylbeta(1-4)Xylbeta(1-4)Xylbeta(1-4)Xylbeta + H2O
2-nitrophenyl beta-D-xylopyranose + Xylbeta(1-4)Xylbeta(1-4)Xylbeta
show the reaction diagram
2-nitrophenyl Xylbeta(1-4)Xylbeta(1-4)Xylbeta(1-4)Xylbeta(1-4)Xylbeta + H2O
2-nitrophenyl Xylbeta(1-4)Xylbeta + Xylbeta(1-4)Xylbeta(1-4)Xyl
show the reaction diagram
2-nitrophenyl Xylbeta(1-4)Xylbeta(1-4)Xylbeta(1-4)Xylbeta(1-4)Xylbeta(1-4)Xylbeta + H2O
2-nitrophenyl Xylbeta(1-4)Xylbeta(1-4)Xylbeta + Xylbeta(1-4)Xylbeta(1-4)Xyl
show the reaction diagram
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the enzyme cleaves the substrate exclusively at the third xylosidic bond from the reducing end
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-
?
2-nitrophenyl xylohexaose + H2O
?
show the reaction diagram
alpha-xylobiosyl fluoride + D-xylose
alpha-xylobiose + HF + D-xylose
show the reaction diagram
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alpha-xylobiosylfluoride + H2O + xylose
alpha-xylobiose + HF + xylose
show the reaction diagram
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presence of xylose is required, some mutants in D263 also produce xylotriose
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?
beta-D-glucopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranose + H2O
?
show the reaction diagram
beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-deoxyxylopyranose + H2O
?
show the reaction diagram
beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-glucopyranose + H2O
?
show the reaction diagram
beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranose + ?
?
show the reaction diagram
beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranose + H2O
beta-D-xylopyranosyl-(1-4)-alpha-D-xylopyranose
show the reaction diagram
beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranose + H2O
?
show the reaction diagram
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-
-
-
?
beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranose + H2O
?
show the reaction diagram
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-
-
-
?
beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranose + H2O
?
show the reaction diagram
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-
-
-
?
oat spelt xylan + H2O
?
show the reaction diagram
soluble oat spelt xylan + H2O
?
show the reaction diagram
wheat flour arabinoxylan + H2O
?
show the reaction diagram
xyloheptaose + H2O
?
show the reaction diagram
xylohexaose + H2O
?
show the reaction diagram
xylohexaose + H2O
xylose + xylobiose
show the reaction diagram
xylononaose + H2O
?
show the reaction diagram
xylooctaose + H2O
?
show the reaction diagram
xylooligosaccharides + H2O
D-xylose + Xylbeta(1-4)Xyl
show the reaction diagram
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-
-
?
xylopentaose + H2O
?
show the reaction diagram
xylopentaose + H2O
xylose + xylobiose
show the reaction diagram
xylopentaose + H2O
xylotetraose + xylobiose
show the reaction diagram
the recombinant enzyme constitutively hydrolyzes a xylose residue from Xn when n is above 2, until the final products are (n-2)X1 and X2
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?
xylotetraose + H2O
?
show the reaction diagram
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?
xylotetraose + H2O
xylose + xylobiose
show the reaction diagram
xylotetraose + H2O
xylotriose + xylobiose
show the reaction diagram
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-
-
?
xylotriose + H2O
?
show the reaction diagram
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?
xylotriose + H2O
D-xylose + xylobiose
show the reaction diagram
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-
-
?
xylotriose + H2O
xylose + xylobiose
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
1-O-fluoro-4-O-alpha-D-xylopyranosyl-beta-D-xylopyranose + H2O
alpha-D-xylopyranose + 1-O-fluoro-beta-D-xylopyranose
show the reaction diagram
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-
-
-
?
additional information
?
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0024 - 0.0344
beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranose
0.005
beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranose
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0.0044
beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranose
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0.0185
beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranose
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6 - 14.8
D-xylose
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
21 - 163
beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranose
162
beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranose
Bacillus halodurans
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73
beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranose
Bacillus halodurans
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175
beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranose
Bacillus halodurans
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0.42 - 11.9
D-xylose
138 - 166
oat spelt xylan
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1099 - 1455
wheat flour arabinoxylan
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
30
2-nitrophenyl Xylbeta(1-4)Xylbeta(1-4)Xylbeta(1-4)Xylbeta(1-4)Xylbeta
Bacillus halodurans
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apparent value, at pH 6.0, 40C
166186
32 - 105
xyloheptaose
10896
5 - 158
xylohexaose
3107
92 - 152
xylononaose
166185
67 - 132
xylooctaose
31926
22
xylopentaose
uncultured bacterium
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apparent value, at pH 6.0, 40C
2425
43
xylotetraose
Bacillus halodurans
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apparent value, at pH 6.0, 40C
1385
65
xylotriose
Bacillus halodurans
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apparent value, at pH 6.0, 40C
1008
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.2 - 7.3
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.3 - 8.2
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pH 5.3: about 60% of maximal activity, pH 8.2: about 50% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35 - 55
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about 50% of maximal activity at 35C and at 55C
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus halodurans (strain ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125)
Bacillus halodurans (strain ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125)
Bacillus halodurans (strain ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125)
Bacillus halodurans (strain ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125)
Bacillus halodurans (strain ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125)
Bacillus halodurans (strain ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125)
Bacillus halodurans (strain ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125)
Bacillus halodurans (strain ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapour-diffusion method using 13.6 mg/ml Rex, 5.6% v/v polyethylene glycol 4000, 70 mM sodium acetate pH 4.6 and 30% v/v glycerol. The crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 52.69, b = 86.02, c = 87.92 A
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mutants of reducing-end xylose-releasing exooligoxylanase with glycosynthase activity, X-ray diffraction structure determination and analysis at 1.39-2.10 A resolution
xylose-enzyme complex is prepared by cocrystallization using a reservoir solution containing 10 mM xylose. The xylobiose complex of the E70A mutant enzyme is prepared by cocrystallization using a reservoir solution containing 10 mM xylotriose
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9.8
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30C, 30 min, stable
659431
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
purified enzyme is stable for at least 4 months at 4C in 25 mM sodium acetate buffer, pH 5.0
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by using a Ni-NTA agarose column
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by using a single-step HisTrap purification performed on a Atka fast protein liquid chromatograph. Protein yield: 140 mg recombinant enzyme per liter cell culture
Ni-NTA affinity column chromatography, HisTrap HP column chromatography, Q Sepharose column chromatography, and SP Sepharose column chromatography
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL-21 Codon-Plus(DE3) RIL by metal affinity chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed as a His-tagged fusion protein in Escherichia coli
expressed in Escherichia coli BL21(DE3)pLysS cells
expression in Escherichia coli
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expression of wild-type and mutant enzymes in Escherichia coli strain BL21
gene rex8A, the gene is located downstream of a GH3 gene (xyl3A), DNA and amino acid sequence determination and analysis, genetic organization, phylogenetic analysis, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL-21 Codon-Plus(DE3) RIL
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D128A
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specific activity is 290fold lower than that of the wild-type enzyme
D263L
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conversion of glycosynthase from converting enzyme, synthesis of some xylotriose from xylobiose + HF + xylose
D263P
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very little glycosynthase or converting activity
D263T
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conversion of glycosynthase from converting enzyme, synthesis of some xylotriose from xylobiose + HF + xylose
D263V
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conversion of glycosynthase from converting enzyme, synthesis of some xylotriose from xylobiose + HF + xylose
D263X
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construction of a D263 mutant library by saturation mutagenesis
D70A
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specific activity is 1545fold lower than that of the wild-type enzyme
H319A
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mutation causes a drastic decrease in activity towards the substrate beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranose
Y198F/D263N
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Y198F/D263N showed 20 times less F- releasing activity than Y198F, which is comparable to that of the corresponding single mutant, D263N. Km: 6.0 (xylose), kcat: 0.42/sec (xylose)
D128A
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specific activity is 290fold lower than that of the wild-type enzyme
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D263A
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specific activity is 4421fold lower than that of the wild-type enzyme
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D70A
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specific activity is 1545fold lower than that of the wild-type enzyme
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H319A
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mutation causes a drastic decrease in activity towards the substrate beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranosyl-(1-4)-beta-D-xylopyranose
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D148A
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site-directed mutagenesis, inactive mutant
D286A
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site-directed mutagenesis, inactive mutant
E90A
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site-directed mutagenesis, inactive mutant
D148A
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site-directed mutagenesis, inactive mutant
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D286A
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site-directed mutagenesis, inactive mutant
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E90A
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site-directed mutagenesis, inactive mutant
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additional information
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method for construction of a mutant engineered reducing-end xylose-releasing exooligoxylanase, with mutation of residues Y198 and D263, that acts as a glycosynthase, reaction mechanism and modelling, overview. Glycosynthases are engineered glycoside hydrolases, that catalyze the synthesis of glycoside from glycosyl-fluoride donors and suitable acceptors, overview
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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a novel method for producing a glycosynthase from an inverting glycoside hydrolase by mutating a residue that holds the nucleophilic water molecule (Y198) with the general base residue while keeping the general base residue intact