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

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EC NUMBER
COMMENTARY hide
3.2.1.63
-
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RECOMMENDED NAME
GeneOntology No.
1,2-alpha-L-fucosidase
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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
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of O-glycosyl bond
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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.
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CAS REGISTRY NUMBER
COMMENTARY hide
37288-45-2
-
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
Bacillus fulminans
-
-
-
Manually annotated by BRENDA team
strain: VIII-210
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-
Manually annotated by BRENDA team
strain: VIII-240
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-
Manually annotated by BRENDA team
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J9UN47
UniProt
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
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
physiological function
-
AfcA plays essential role in degrading alpha1,2-fucosylated milk oligosaccharides and also glycoconjugates, in the gastrointestinal tracts of the host
additional information
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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
-
-
-
-
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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
J9UN47
-
-
-
?
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
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i.e. lacto-N-fucopentaose I
-
?
beta-L-fucosyl fluoride + lactose + H2O
?
show the reaction diagram
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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
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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
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i.e. 2'-fucosyllactose
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-
?
Galbeta(1-4)(Fucalpha(1-3))Glc + H2O
?
show the reaction diagram
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i.e. 3-fucosyllactose, at 0.85% of the activity with 2'-fucosyllactose
-
-
?
lacto-N-fucopentaose I
?
show the reaction diagram
Bacillus fulminans
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-
-
-
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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
B2C4H2
-
-
-
?
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
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at 33% of the activity with 2'-fucosyllactose
-
-
?
submaxillary glycoprotein + H2O
L-fucose + ?
show the reaction diagram
-
degradation of blood group substances
-
?
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
2'-fucosyllactose + H2O
L-fucose + lactose
show the reaction diagram
-
-
-
-
?
submaxillary glycoprotein + H2O
L-fucose + ?
show the reaction diagram
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degradation of blood group substances
-
?
additional information
?
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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
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-
-
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
CaCl2
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slight activation
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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
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1 mM, 27% inhibition
D-mannose
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10 mM, 22% inhibition
deoxycholate
Bacillus fulminans
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deoxyfuconojirimycin
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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
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L-fucose
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10 mM, 91% inhibition
lactose
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10 mM, 27% inhibition
MgCl2
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Mn2+
Bacillus fulminans
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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
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
maltose
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10 mM, 10% activation
NEM
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1 mM, 20% activation
Sodium azide
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for D766A mutant only
additional information
B2C4H2
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
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-
0.035 - 17
2-fucosyllactose
2
alpha-Fuc-(1-2)-beta-Gal-(1-3)-beta-GlcNAc-(1-3)-beta-Gal-(1-4-)-beta-Glc
Bacillus fulminans
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-
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1.6
glycopeptide desialyzed porcine submaxillary mucin
Bacillus fulminans
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-
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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
J9UN47
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
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00186
-
-
0.12
Bacillus fulminans
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-
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5
J9UN47
assay at
5 - 6.5
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-
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2.4 - 7.5
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5 - 8
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pH 5.0: about 60% of maximal activity, pH 8.0: about 35% of maximal activity
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 60
-
20°C: about 45% of maximal activity, 60°C: about 45% of maximal activity
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3
-
isoelectric focusing
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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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
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commercial preparation
Manually annotated by BRENDA team
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70000 - 80000
Bacillus fulminans
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gel filtration
81000
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2 * 81000, SDS-PAGE
196000
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gel filtration
200000
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value greater than, multiple isozymic forms
205000
x * 205000, calculated from sequence
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 205000, calculated from sequence
dimer
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2 * 81000, SDS-PAGE
monomer
J9UN47
crystal structure analysis, solution small angle x-ray scattering
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POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
J9UN47
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
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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, 20°C, 2-3 days, X-ray diffraction structure determination and analysis at 1.38-156 A resolution, molecular replacement method
J9UN47
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9
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37°C, 6 h, stable
665770
6.5 - 7.5
-
Fuc domain is stable for 12 h
665375, 665768
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35
30 min, Fuc domain is stable below
70
Bacillus fulminans
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5 min, 77% loss of activity
80
Bacillus fulminans
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5 min, complete loss of activity
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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
-
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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
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partial
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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
J9UN47
wild-type and mutant derivatives of the catalytic domain of AfcA (Fuc domain)
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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
J9UN47
wild-type and mutant derivatives of the catalytic domain of AfcA (Fuc domain)
-
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D766A
-
site directed mutagenesis
D766E
-
site directed mutagenesis
D766G
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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
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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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
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LINKS TO OTHER DATABASES (specific for EC-Number 3.2.1.63)
ExplorEnz
ExPASy
KEGG
MetaCyc
SABIO-RK
NCBI: PubMed, Protein, Nucleotide, Structure, Genome, OMIM
IUBMB Enzyme Nomenclature
PROSITE Database of protein families and domains
InterPro (database of protein families, domains and functional sites)