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

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The enzyme appears in viruses and cellular organisms

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
gene lnpA, located in a gene cluster encoding a specific pathway for utilization of galacto-N-biose and lacto-N-biose
UniProt
Manually annotated by BRENDA team
-
Q0TTN3
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
strain JCM6425
UniProt
Manually annotated by BRENDA team
strain JCM6473
UniProt
Manually annotated by BRENDA team
strain CMCP6LNBP
Q8D536
UniProt
Manually annotated by BRENDA team
strain CMCP6LNBP
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
493
0.22 - 0.73
beta-D-galactopyranosyl-1,3-N-acetyl-D-galactosamine
11582
0.01 - 13
beta-D-galactopyranosyl-1,3-N-acetyl-D-glucosamine
10116
0.031 - 8.5
galacto-N-biose
4122
0.34 - 9.3
lacto-N-biose I
3718
3.9
N-acetyl-D-galactosamine
Propionibacterium acnes
B7XEJ0, B7XEJ1
in 100 mM MOPS buffer (pH 7.0) at 37C
1075
0.044
N-acetyl-D-glucosamine
Propionibacterium acnes
B7XEJ0, B7XEJ1
in 100 mM MOPS buffer (pH 7.0) at 37C
300
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
-
pH 7.0, 30C
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
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 30C
30 - 55
Q8D536
the enzyme is stable at temperatures up to 30C during incubation for 30 min, the reactions proceed linearly for 5 min at temperatures up to 37C but do not proceed linearly at 45C, no reaction is observed at 55C
37
the enzyme is stable up to 37C
45
protein is thermostable up to 45C
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)
-
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
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