Information on EC 2.4.1.211 - 1,3-beta-galactosyl-N-acetylhexosamine phosphorylase

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

EC NUMBER
COMMENTARY hide
2.4.1.211
-
RECOMMENDED NAME
GeneOntology No.
1,3-beta-galactosyl-N-acetylhexosamine phosphorylase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
beta-D-galactopyranosyl-(1->3)-N-acetyl-D-glucosamine + phosphate = alpha-D-galactopyranose 1-phosphate + N-acetyl-D-glucosamine
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexosyl group transfer
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
beta-D-galactopyranosyl-(1->3)-N-acetyl-D-hexosamine:phosphate galactosyltransferase
Reaction also occurs with beta-D-galactopyranosyl-(1->3)-N-acetyl-D-galactosamine as the substrate, giving N-acetyl-D-galactosamine as the product.
CAS REGISTRY NUMBER
COMMENTARY hide
224427-06-9
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
Q0TTN3
UniProt
Manually annotated by BRENDA team
strain JCM6425
UniProt
Manually annotated by BRENDA team
strain JCM6473
UniProt
Manually annotated by BRENDA team
no activity in Bifidobacterium adolescentis
-
-
-
Manually annotated by BRENDA team
no activity in Bifidobacterium angulatum
-
-
-
Manually annotated by BRENDA team
no activity in Bifidobacterium animalis subsp. lactis
-
-
-
Manually annotated by BRENDA team
no activity in Bifidobacterium catenulatum
-
-
-
Manually annotated by BRENDA team
no activity in Bifidobacterium dentium
-
-
-
Manually annotated by BRENDA team
no activity in Bifidobacterium pseudocatenulatum
-
-
-
Manually annotated by BRENDA team
no activity in Bifidobacterium thermophilum
-
-
-
Manually annotated by BRENDA team
-
Q8D536
UniProt
Manually annotated by BRENDA team
-
Q8D536
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
-
the GalHexNAcP belongs to the glycoside hydrolase family 112, GH112
metabolism
physiological function
additional information
-
the residues at positions 162, 161, and 336 determine the substrate specificity, structure-function relationship, molecular docking, and specificity prediction, overview. The side-chain hydroxyl group of S336 forms a hydrogen bond with the side-chain nitrogen atom of R358, which plays a significant role in catalysis
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
alpha-D-galactose 1-phosphate + N-acetyl-galactosamine
beta-D-galactopyranosyl-1,3-N-acetyl-D-galactosmine + phosphate
show the reaction diagram
beta D-galactosido-(1,3)-N-acetylgalactosamine + H2O
alpha-D-galactose-1-phosphate + GalNAc
show the reaction diagram
-
-
-
-
r
beta-D-galactopyranosyl-1,3-N-acetyl-D-galactosamine + phosphate
alpha-D-galactopyranose 1-phosphate + N-acetyl-D-galactosamine
show the reaction diagram
beta-D-galactopyranosyl-1,3-N-acetyl-D-galactosamine + phosphate
alpha-D-galactose 1-phosphate + N-acetyl-D-galactosamine
show the reaction diagram
beta-D-galactopyranosyl-1,3-N-acetyl-D-galactosmine + phosphate
alpha-D-galactose 1-phosphate + N-acetyl-D-galactosamine
show the reaction diagram
beta-D-galactopyranosyl-1,3-N-acetyl-D-galactosmine + phosphate
alpha-D-galactose 1-phosphate + N-acetyl-galactosamine
show the reaction diagram
beta-D-galactopyranosyl-1,3-N-acetyl-D-glucosamine + phosphate
alpha-D-galactose 1-phosphate + N-acetyl-D-glucosamine
show the reaction diagram
beta-D-galactopyranosyl-1,3-N-acetyl-D-glucosamine + phosphate
alpha-D-galactose 1-phosphate + N-acetyl-glucosamine
show the reaction diagram
beta-D-galactopyranosyl-1,3-N-acetyl-D-glucosoamine + phosphate
alpha-D-galactopyranose 1-phosphate + N-acetyl-D-glucosamine
show the reaction diagram
galacto-N-biose + phosphate
alpha-D-galactose 1-phosphate + N-acetyl-D-galactosamine
show the reaction diagram
lacto-N-biose I + phosphate
alpha-D-galactose 1-phosphate + N-acetyl-D-glucosamine
show the reaction diagram
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
additional information
?
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.72 - 24
alpha-D-galactose 1-phosphate
10 - 140
beta-D-galactopyranosyl-1,3-N-acetyl-D-galactosamine
1.7 - 5.6
beta-D-galactopyranosyl-1,3-N-acetyl-D-galactosmine
2.1 - 1300
beta-D-galactopyranosyl-1,3-N-acetyl-D-glucosamine
6.7 - 120
galacto-N-biose
1.6
galactose-1-phosphate
-
-
2.8 - 46
lacto-N-biose I
3.3 - 11
N-acetyl-D-galactosamine
1.9 - 130
N-acetyl-D-glucosamine
0.064 - 2.7
phosphate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.09 - 10.7
alpha-D-galactose 1-phosphate
2.2 - 103
beta-D-galactopyranosyl-1,3-N-acetyl-D-galactosamine
14 - 45
beta-D-galactopyranosyl-1,3-N-acetyl-D-glucosamine
3.5 - 65
galacto-N-biose
15 - 40
lacto-N-biose I
34 - 48
N-acetyl-D-galactosamine
5.9 - 49
N-acetyl-D-glucosamine
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.005 - 6.21
alpha-D-galactose 1-phosphate
0.22 - 0.73
beta-D-galactopyranosyl-1,3-N-acetyl-D-galactosamine
0.01 - 13
beta-D-galactopyranosyl-1,3-N-acetyl-D-glucosamine
0.031 - 8.5
galacto-N-biose
0.34 - 9.3
lacto-N-biose I
3.9
N-acetyl-D-galactosamine
in 100 mM MOPS buffer (pH 7.0) at 37°C
0.044
N-acetyl-D-glucosamine
in 100 mM MOPS buffer (pH 7.0) at 37°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0002
-
D313N, no activity, D313 is probably the catalytic proton donor site
3.5
-
alpha-D-galactose 1-phosphate as substrate
10.5
-
LNBP1, all mutants display decreased activities, D313N has no activity
19
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pH 7.0, 30°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 6
-
reverse reaction
6 - 6.5
-
galactophosphorolytic activity
6 - 7
optimum activity at neutral pH; optimum activity at neutral pH
6.5 - 7.5
Q8D536
-
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 5.5
Q0TTN3
in presence of sodium acetate
5.5 - 6.5
Q0TTN3
in presence of MES-NaOH
6.5 - 7.5
Q0TTN3
in presence of Mops-NaOH
7.5 - 9
Q0TTN3
in presence of Tris-HCl
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50 - 55
-
phosphorolysis and reverse reaction
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 60
temperature-activity profiles of recombinant wild-type and mutant enzymes, overview
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
proteins are predicted not to possess N-terminal signal peptides; proteins are predicted not to possess N-terminal signal peptides
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bifidobacterium longum subsp. longum (strain ATCC 15707 / DSM 20219 / JCM 1217 / NCTC 11818 / E194b)
Bifidobacterium longum subsp. longum (strain ATCC 15707 / DSM 20219 / JCM 1217 / NCTC 11818 / E194b)
Bifidobacterium longum subsp. longum (strain ATCC 15707 / DSM 20219 / JCM 1217 / NCTC 11818 / E194b)
Bifidobacterium longum subsp. longum (strain ATCC 15707 / DSM 20219 / JCM 1217 / NCTC 11818 / E194b)
Bifidobacterium longum subsp. longum (strain ATCC 15707 / DSM 20219 / JCM 1217 / NCTC 11818 / E194b)
Bifidobacterium longum subsp. longum (strain ATCC 15707 / DSM 20219 / JCM 1217 / NCTC 11818 / E194b)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
83480
His-tagged enzyme, calculated from amino acid sequence
83580
Q8D536
calculated from amino acid sequence
84327
-
x * 84327, calculated
86000
x * 86000, SDS-PAGE
140000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
N-terminal amino acid sequence
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in complex with beta-D-galactopyranosyl-1,3-N-acetyl-D-glucosamine and beta-D-galactopyranosyl-1,3-N-acetyl-D-galactosamine
sitting drop vapor diffusion method, using 0.1 M sodium cacodylate (pH 6.5), 0.2 M Mg(NO3)2, and 15% (v/v) polyethylene glycol 4000
-
purified recombinant enzyme mutant C236Y, X-ray diffraction structure determination and analysis at 2.6 A resolution
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 6
Q0TTN3
in presence of sodium acetate
684635
6 - 7
Q0TTN3
in presence of MES-NaOH
684635
6.5 - 7.5
6.5 - 9
Q8D536
-
701782
7.5 - 9
Q0TTN3
in presence of Tris-HCl
684635
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 50
Q0TTN3
in 100 mM buffer containing 20 micromolar DTT
30
protein is thermostable up to 30°C
30 - 55
Q8D536
the enzyme is stable at temperatures up to 30°C during incubation for 30 min, the reactions proceed linearly for 5 min at temperatures up to 37°C but do not proceed linearly at 45°C, no reaction is observed at 55°C
37
the enzyme is stable up to 37°C
45
protein is thermostable up to 45°C
55
rapid inactivation of wild-type enzyme, slower inactivation of mutants R209H, G437S, and N506S, mutants C236Y and D576V retain some activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
unstable after cell lysis
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-NTA agarose column chromatography
of the recombinant protein by Ni-nitrilotriacetic acid agarose column chromatography
Q0TTN3
of the recombinant protein by Ni-NTA spin column chromatography
-
of the recombinant proteins by Ni-NTA spin column chromatography
-
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, sequence comparisons, phylogenetic analysis, exxpression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 (DE3)
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expressed in Escherichia coli B834 (DE3) cells
expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli BL21(DE3); expressed in Escherichia coli BL21(DE3)
expression in Escherichia coli
gene lnpA, sequence comparisons, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain strain BL21
the non-labeled enzyme is expressed in Escherichia coli BL21(DE3) cells, the selenomethionine-labeled enzyme is expressed in Escherichia coli strain B834 (DE3)
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D108N
-
site-directed mutagenesis
D160N
-
site-directed mutagenesis
D182N
-
site-directed mutagenesis
D232N
-
site-directed mutagenesis
D260N
-
site-directed mutagenesis
D280N
-
site-directed mutagenesis
D29N
-
site-directed mutagenesis
D313N
-
site-directed mutagenesis
D331N
-
site-directed mutagenesis
D344N
-
site-directed mutagenesis
D348N
-
site-directed mutagenesis
D35N
-
site-directed mutagenesis
D373N
-
site-directed mutagenesis
D490N
-
site-directed mutagenesis
D491N
-
site-directed mutagenesis
D561N
-
site-directed mutagenesis
E249Q
-
site-directed mutagenesis
E259Q
-
site-directed mutagenesis
E319Q
-
site-directed mutagenesis
E356Q
-
site-directed mutagenesis
E377Q
-
site-directed mutagenesis
E475Q
-
site-directed mutagenesis
E568Q
-
site-directed mutagenesis
E691Q
-
site-directed mutagenesis
D313N
-
mutant with undetectable activity
F364N
-
mutant shows severely impaired activity
N166A
-
mutant shows severely impaired activity
P161S
-
site-directed mutagenesis, the mutation leads to an increase in the selectivity on lacto-N-biose I
P161S/S336A
-
site-directed mutagenesis
R210E
-
the mutant shows no detectable activity
R32E
-
the mutant shows no detectable activity
R358E
-
the mutant shows no detectable activity
S336A
-
site-directed mutagenesis, the mutation leads to an increase in the selectivity on lacto-N-biose I
V162T
-
site-directed mutagenesis, the mutation leads to an increase in the selectivity on galacto-N-biose
Y362F
-
the mutation results in about 1000fold reduction of the catalytic efficiency
Y362N
-
the mutation results in the complete loss of the activity
C236E
random mutagenesis, the mutant shows decreased thermostability and reduced activity compared to the wild-type
C236F
random mutagenesis, the mutant shows highly increased thermostability and inacreased activity compared to the wild-type
C236H
random mutagenesis, the mutant shows slightly increased thermostability compared to the wild-type
C236P
random mutagenesis, the mutant shows highly increased thermostability and reduced activity compared to the wild-type
C236W
random mutagenesis, the mutant shows slightly increased thermostability compared to the wild-type
C236Y
random mutagenesis, the mutant shows highly increased thermostability and increased activity compared to the wild-type. In the mutant, the hydroxyl group of Tyr236 forms a hydrogen bond with the carboxyl group of E319. Mutant C236Y shows a 1.6fold higher specific activity than the wild-type
C236Y/D576V
random mutagenesis, the mutant shows highly increased thermostability compared to the wild-type
D576A
random mutagenesis, the mutant shows highly increased thermostability compared to the wild-type
D576F
random mutagenesis, the mutant shows highly increased thermostability and reduced activity compared to the wild-type
D576G
random mutagenesis, the mutant shows highly increased thermostability and increased activity compared to the wild-type
D576I
random mutagenesis, the mutant shows highly increased thermostability compared to the wild-type
D576L
random mutagenesis, the mutant shows highly increased thermostability compared to the wild-type
D576M
random mutagenesis, the mutant shows highly increased thermostability compared to the wild-type
D576P
random mutagenesis, the mutant shows decreased thermostability compared to the wild-type
D576V
random mutagenesis, the mutant shows highly increased thermostability compared to the wild-type
D576W
random mutagenesis, the mutant shows highly increased thermostability and reduced activity compared to the wild-type
G437S
random mutagenesis, the mutant shows slightly increased thermostability and slightly reducd activity compared to the wild-type
N506S
random mutagenesis, the mutant shows slightly increased thermostability and reduced activity compared to the wild-type
R290H
random mutagenesis, the mutant shows slightly increased thermostability and slightly reduced activity compared to the wild-type
R290H/G437S/N506S
random mutagenesis, the mutant shows highly increased thermostability and reduced activity compared to the wild-type
C236Y
-
random mutagenesis, the mutant shows highly increased thermostability and increased activity compared to the wild-type. In the mutant, the hydroxyl group of Tyr236 forms a hydrogen bond with the carboxyl group of E319. Mutant C236Y shows a 1.6fold higher specific activity than the wild-type
-
D576A
-
random mutagenesis, the mutant shows highly increased thermostability compared to the wild-type
-
D576V
-
random mutagenesis, the mutant shows highly increased thermostability compared to the wild-type
-
R290H
-
random mutagenesis, the mutant shows slightly increased thermostability and slightly reduced activity compared to the wild-type
-
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
-
LNBP is cultured with sucrose phosphorylase, UDP-glucose hexose 1-phosphate uridylyltransferase, and UDP-glucose 4-epimerase to produce beta-D-galactopyranosyl-1,3-N-acetyl-D-glucosamine using sucrose as substrate in a 10l reaction mixture, 500 mmol beta-D-galactopyranosyl-1,3-N-acetyl-D-glucosamine are produced after 600 h, beta-D-galactopyranosyl-1,3-N-acetyl-D-glucosamine can be used as bifidus factor in human milk
medicine
-
galactosyl-N-acetylhexosamine phosphorylase has a predominant role in the digestive tract of human and in the metabolism of galactose
nutrition
-
dairy industry production of different fermented bifidobacteria milks
synthesis
Show AA Sequence (209 entries)
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