Information on EC 3.2.1.63 - 1,2-alpha-L-fucosidase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
3.2.1.63
-
RECOMMENDED NAME
GeneOntology No.
1,2-alpha-L-fucosidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
hydrolysis of (1->4)-alpha-D-glucosidic linkages in amylaceous polysaccharides, to remove successive maltotetraose residues from the non-reducing chain ends
show the reaction diagram
methyl-2-alpha-L-fucopyranosyl-beta-D-galactoside + H2O = L-fucose + methyl beta-D-galactoside
show the reaction diagram
; high specificity for non-reducing terminal L-fucose residues alpha 1,2-linked to D-galactose residues
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of O-glycosyl bond
SYSTEMATIC NAME
IUBMB Comments
2-alpha-L-fucopyranosyl-beta-D-galactoside fucohydrolase
Highly specific for non-reducing terminal L-fucose residues linked to D-galactose residues by a 1,2-alpha-linkage. Not identical with EC 3.2.1.111 1,3-alpha-L-fucosidase.
CAS REGISTRY NUMBER
COMMENTARY hide
37288-45-2
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Bacillus fulminans
-
-
-
Manually annotated by BRENDA team
strain: VIII-210
-
-
Manually annotated by BRENDA team
strain: VIII-240
-
-
Manually annotated by BRENDA team
no activity in Bifidobacterium longum
-
-
-
Manually annotated by BRENDA team
almond
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-
Manually annotated by BRENDA team
i.e. Ruminococcus AB strain VI-268, ATCC35913
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-
Manually annotated by BRENDA team
strains: VIII-239 and IX-70
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-
Manually annotated by BRENDA team
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GenBank
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
physiological function
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
2'-fucosyllactose + H2O
?
show the reaction diagram
-
hydrolysis by the Fuc domain of AfcA. N423 and D766 are critical for activating an attacking water molecule and N421 is critical for maintaining the water molecule and the side-chain of E566 (a general acid) in their proper orientations
-
-
?
2'-fucosyllactose + H2O
fucose + lactose
show the reaction diagram
2'-fucosyllactose + H2O
L-fucose + lactose
show the reaction diagram
2'-L-fucosyllactose + H2O
L-fucose + lactose
show the reaction diagram
2-fucosyllactitol + H2O
fucose + lactitol
show the reaction diagram
-
-
-
-
-
2-O-alpha-L-fucopyranosyl-D-galactose + H2O
L-fucose + galactose
show the reaction diagram
4-nitrophenyl-alpha-L-fucoside + H2O
4-nitrophenol + L-fucose
show the reaction diagram
-
-
-
?
alpha-Fuc-(1-2)-beta-Gal-(1-3)-beta-GlcNAc-(1-3)-beta-Gal-(1-4)-beta-Glu + H2O
alpha-L-fucose + beta-Gal-(1-3)-beta-GlcNAc-(1-3)-beta-Gal-(1-4)-beta-Glu
show the reaction diagram
-
i.e. lacto-N-fucopentaose I
-
?
beta-L-fucosyl fluoride + lactose + H2O
?
show the reaction diagram
-
Hehre resynthesis-hydrolysis is catalyzed by the wild-type Fuc domain of AfcA. Conversion of the inverting alpha-glycosidase to a glycosynthase
-
-
?
blood group substance H + H2O
?
show the reaction diagram
Fucalpha(1-2)Galbeta(1-3)GlcNAcbeta(1-3)Galbeta(1-4)Glc + H2O
L-fucose + Galbeta(1-3)GlcNAcbeta(1-3)Galbeta(1-4)Glc
show the reaction diagram
-
i.e. lacto-N-fucopentaose I, at 29% of the activity with 2'-fucosyllactose
-
-
?
Fucalpha(1-2)Galbeta(1-4)Glc + H2O
L-fucose + Galbeta(1-4)Glc
show the reaction diagram
-
i.e. 2'-fucosyllactose
-
-
?
Galbeta(1-4)(Fucalpha(1-3))Glc + H2O
?
show the reaction diagram
-
i.e. 3-fucosyllactose, at 0.85% of the activity with 2'-fucosyllactose
-
-
?
lacto-N-fucopentaose I
?
show the reaction diagram
Bacillus fulminans
-
-
-
-
-
lacto-N-fucopentaose I + H2O
?
show the reaction diagram
methyl-2-alpha-L-fucopyranosyl-beta-D-galactoside + H2O
L-fucose + methyl beta-D-galactoside
show the reaction diagram
mucin + H2O
?
show the reaction diagram
-
-
-
?
mucin + H2O
L-fucose + ?
show the reaction diagram
O-alpha-L-fucose-(1-2)-O-beta-D-galactose-(1-4)-D-glucose + H2O
L-fucose + lactose
show the reaction diagram
porcine gastric mucin + H2O
?
show the reaction diagram
-
at 33% of the activity with 2'-fucosyllactose
-
-
?
submaxillary glycoprotein + H2O
L-fucose + ?
show the reaction diagram
-
degradation of blood group substances
-
?
additional information
?
-
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
2'-fucosyllactose + H2O
L-fucose + lactose
show the reaction diagram
submaxillary glycoprotein + H2O
L-fucose + ?
show the reaction diagram
-
degradation of blood group substances
-
?
additional information
?
-
-
inductively produced in culture medium containing porcine gastric mucin, weakly induced by L-fucose and D-arabinose, but not by other sugars including D-glucose
-
-
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
CaCl2
-
slight activation
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(2R,3R,4R,5R,6S)-2-(aminomethyl)-6-methylpiperidine-3,4,5-triol
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(2R,3S,4S,5R,6S)-2-benzyl-6-methylpiperidine-3,4,5-triol
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(2S,3R,4S,5S,6R)-2-methyl-6-(propan-2-yl)piperidine-3,4,5-triol
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2-(1H-indol-3-yl)-N-[[(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-methylpiperidin-2-yl]methyl]acetamide
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2-cyclopentyl-2-phenyl-N-[[(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-methylpiperidin-2-yl]methyl]acetamide
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3-(1H-indol-3-yl)-N-[[(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-methylpiperidin-2-yl]methyl]propanamide
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9-oxo-N-[[(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-methylpiperidin-2-yl]methyl]-9H-fluorene-1-carboxamide
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BaCl2
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1 mM, 63% inhibition
CaCl2
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1 mM, 60% inhibition
CoCl2
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1 mM, 38% inhibition
CuSO4
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1 mM, 86% inhibition
cysteine
-
1 mM, 27% inhibition
D-mannose
-
10 mM, 22% inhibition
deoxycholate
Bacillus fulminans
-
-
deoxyfuconojirimycin
-
-
dithiothreitol
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1 mM, 22% inhibition
FeSO4
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1 mM, complete inhibition
glutathione
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1 mM, 21% inhibition
iodoacetamide
-
-
L-fucose
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10 mM, 91% inhibition
lactose
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10 mM, 27% inhibition
MgCl2
-
-
Mn2+
Bacillus fulminans
-
-
N-[[(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-methylpiperidin-2-yl]methyl]-1-benzofuran-2-carboxamide
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N-[[(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-methylpiperidin-2-yl]methyl]-1H-indole-2-carboxamide
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N-[[(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-methylpiperidin-2-yl]methyl]acetamide
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NEM
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0.1 mM, 24% inhibition
NiSO4
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1 mM, 59% inhibition
p-chloromercuribenzoate
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-
p-Chloromercuriphenylsulfonate
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1 mM, 12% inhibition
p-mercuribenzoate
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1 mM, 23% inhibition
SDS
Bacillus fulminans
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-
ZnCl2
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1 mM, 43% inhibition
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
maltose
-
10 mM, 10% activation
NEM
-
1 mM, 20% activation
Sodium azide
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for D766A mutant only
additional information
mucin highly induces gene expression
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.53 - 2.5
2'-fucosyllactose
0.675
2-fucosyllactitol
-
-
0.035 - 17
2-fucosyllactose
2
alpha-Fuc-(1-2)-beta-Gal-(1-3)-beta-GlcNAc-(1-3)-beta-Gal-(1-4-)-beta-Glu
Bacillus fulminans
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-
-
1.6
glycopeptide desialyzed porcine submaxillary mucin
Bacillus fulminans
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-
-
0.175
glycoprotein
Bacillus fulminans
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hog, submaxillary
0.083
methyl 2-O-alpha-L-fucopyranosyl-beta-D-galactoside
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-
0.33
porcine submaxillary mucin
Bacillus fulminans
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-
-
additional information
additional information
steady-state kinetics of deglycosylated and glycosylated enzyme, overview
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0000163
(2R,3R,4R,5R,6S)-2-(aminomethyl)-6-methylpiperidine-3,4,5-triol
pH and temperature not specified in the publication
0.005
(2R,3S,4S,5R,6S)-2-benzyl-6-methylpiperidine-3,4,5-triol
pH and temperature not specified in the publication
0.006
(2S,3R,4S,5S,6R)-2-methyl-6-(propan-2-yl)piperidine-3,4,5-triol
pH and temperature not specified in the publication
0.000000475
2-(1H-indol-3-yl)-N-[[(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-methylpiperidin-2-yl]methyl]acetamide
pH and temperature not specified in the publication
0.001005
2-cyclopentyl-2-phenyl-N-[[(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-methylpiperidin-2-yl]methyl]acetamide
pH and temperature not specified in the publication
0.000000105
3-(1H-indol-3-yl)-N-[[(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-methylpiperidin-2-yl]methyl]propanamide
pH and temperature not specified in the publication
0.000000441
N-[[(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-methylpiperidin-2-yl]methyl]-1-benzofuran-2-carboxamide
pH and temperature not specified in the publication
0.000000427
N-[[(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-methylpiperidin-2-yl]methyl]-1H-indole-2-carboxamide
pH and temperature not specified in the publication
0.00000447
N-[[(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-methylpiperidin-2-yl]methyl]acetamide
pH and temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00186
-
-
0.12
Bacillus fulminans
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-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 6.5
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-
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2.4 - 7.5
-
-
5 - 8
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pH 5.0: about 60% of maximal activity, pH 8.0: about 35% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 60
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20C: about 45% of maximal activity, 60C: about 45% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3
-
isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
inductively produced in culture medium containing porcine gastric mucin, weakly induced by L-fucose and D-arabinose, but not by other sugars including glucose
Manually annotated by BRENDA team
additional information
-
commercial preparation
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70000 - 80000
Bacillus fulminans
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gel filtration
81000
-
2 * 81000, SDS-PAGE
196000
-
gel filtration
200000
-
value greater than, multiple isozymic forms
205000
x * 205000, calculated from sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
-
2 * 81000, SDS-PAGE
monomer
crystal structure analysis, solution small angle x-ray scattering
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
prediction of three N-glycosylation sites on Asn187, Asn280, and Asn401 (residues numbered for the protein without signal peptide). Deglycosylation of the recombinant enzyme expressed from Pichia pastoris by treatment with Endo H or peptide N-glycosidase F. The N-glycans on Asn-280 of the TIM barrel domain retained a chitobiose core and high mannose type substitutions, GlcNAc-beta1-4GlcNAc-beta1-4(Man-alpha1-6)Man-alpha1-3Man-alpha1-2Man-alpha1-2Man, possibly protected from EndoH by the beta,gamma-crystallin domain. The other two N-glycosylation sites Asn187 and Asn401 are each attached to one GlcNAc residue, indicative of Endo H cleavage. No effect of Endo H deglycosylation on the catalytic efficiency with small fucosylated substrates or on thermal stability, but higher solubility of the glycosylated than the deglycosylated enzyme
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
of the AfcA catalytic domain in unliganded and complexed forms with the inhibitor deoxyfuconojirimycin, the substrate 2-fucosyllactose, and the products L-fucose and lactose at 1.12-2.10 A resolution
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purified deglycosylated recombinant enzyme in open or closed form or complexed with L-fucose, sitting-drop vapor diffusion method or batch method, several different crystal forms, mixing 0.001 ml of 14-16 mg/ml Endo H-treated fucosidase in 25 mM Tris, pH 7.5, with 0.001 ml of precipitant solution containing 0.1 M Tris, pH 8.0, and either 30% or 40% w/v PEG MME 2000, 20C, 2-3 days, X-ray diffraction structure determination and analysis at 1.38-156 A resolution, molecular replacement method
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9
-
37C, 6 h, stable
665770
6.5 - 7.5
-
Fuc domain is stable for 12 h
665375, 665768
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35
30 min, Fuc domain is stable below
70
Bacillus fulminans
-
5 min, 77% loss of activity
80
Bacillus fulminans
-
5 min, complete loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
dialysis, water: rapid loss of activity, buffers: precipitation with considerable inactivation below pH 5.5
-
lyophilization causes inactivation
-
repeated freezing and thawing causes inactivation
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
of the recombinant protein by saturated ammonium sulfate precipitation and Q sepharose fast flow, CHT ceramic hydroxyapatite and gel filtration column chromatography
-
partial
-
recombinant enzyme from Pichia pastoris by gel filtration and anion exchange chromatography, recombinant His6-tagged and MBP-fused crystallin domain from Escherichia coli by nickel affinity chromatography
wild-type and mutant derivatives of the catalytic domain of AfcA (Fuc domain)
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression of 1,2-alpha-L-fucosidase in Escherichia coli, expression of the Fuc domain (responsible for fucosidase activity) as a hexahistidine-tagged protein in Escherichia coli
gene afcA, expression in Escherichia coli and in enzyme-deficient Bifidobacterium longum strain 105-A which renders the transformant able to utilize 2'-fucosyllactose as the sole carbon source
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overexpression in Escherichia coli, selenomethionine-substituted protein
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sequence comparisons, recombinant enzyme expression of the enzyme including its native signal peptide from pPICZAalpha without the C-terminal Myc/His6 tags in Pichia pastoris, expression of the His6-tagged crystallin domain, residues 501-585, of the enzyme fused to maltose-binding protein at the N-terminus in Escherichia coli
wild-type and mutant derivatives of the catalytic domain of AfcA (Fuc domain)
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D766A
-
site directed mutagenesis
D766E
-
site directed mutagenesis
D766G
-
most effectively synthesizes 2'-fucosyllactose
E485A
-
site directed mutagenesis
E566A
-
site directed mutagenesis
N421A
-
site directed mutagenesis
N421G/N423G
-
site directed mutagenesis
N423D
-
site directed mutagenesis
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
-
1,2-alpha-L-fucosynthase, derived from an inverting alpha-glycosidase (AfcA) and from a glycosidase with an unusual reaction mechanism, may serve as a promising tool to create biologically active compounds that can be used not only for prebiotics but also for clinical treatments aimed to regulate various cellular processes and infectious diseases