Information on EC 2.4.1.62 - ganglioside galactosyltransferase

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

EC NUMBER
COMMENTARY hide
2.4.1.62
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RECOMMENDED NAME
GeneOntology No.
ganglioside galactosyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
UDP-alpha-D-galactose + an N-acetyl-beta-D-galactosaminyl-(1->4)-[alpha-N-acetylneuraminyl-(2->3)]-beta-D-galactosyl-(1->4)-beta-D-glucosyl-(1<->1)-ceramide = UDP + a beta-D-galactosyl-(1->3)-N-acetyl-beta-D-galactosaminyl-(1->4)-[alpha-N-acetylneuraminyl-(2->3)]-beta-D-galactosyl-(1->4)-beta-D-glucosyl-(1<->1)-ceramide
show the reaction diagram
The substrate is known as GM2.
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexosyl group transfer
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Other types of O-glycan biosynthesis
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Glycosphingolipid biosynthesis - ganglio series
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Metabolic pathways
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SYSTEMATIC NAME
IUBMB Comments
UDP-alpha-D-galactose:N-acetyl-beta-D-galactosaminyl-(1->4)-[alpha-N-acetylneuraminyl-(2->3)]-beta-D-galactosyl-(1->4)-beta-D-glucosyl-(1<->1)-ceramide 3-beta-D-galactosyltransferase
The substrate is also known as gangloside GM2, the product as gangloside GM1a
CAS REGISTRY NUMBER
COMMENTARY hide
37217-28-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
embryonic
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Manually annotated by BRENDA team
no activity in Mesocricetus auratus
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-
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Manually annotated by BRENDA team
frog
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Manually annotated by BRENDA team
Sprague-Dawley
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-
Manually annotated by BRENDA team
Wistar
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Manually annotated by BRENDA team
fetus
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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Cgt deficient mice, no accumulation of galactolipids, increased ceramide levels
physiological function
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post-translationally regulated by ER-associated degradation involving sigma-1 receptor chaperones
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
UDP-alpha-D-galactose + BSA-conjugated C6-ceramide
UDP + ?
show the reaction diagram
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-
-
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?
UDP-Gal + GA2-FCHASE
?
show the reaction diagram
FCHASE: 6-(fluorescein-5-carboxamido)hexanoic acid succinimidyl ester
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-
?
UDP-Gal + GalNAcalpha-FCHASE
?
show the reaction diagram
FCHASE: 6-(fluorescein-5-carboxamido)hexanoic acid succinimidyl ester
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-
?
UDP-Gal + GalNAcbeta-FCHASE
?
show the reaction diagram
FCHASE: 6-(fluorescein-5-carboxamido)hexanoic acid succinimidyl ester
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-
?
UDP-Gal + GM2-FCHASE
?
show the reaction diagram
FCHASE: 6-(fluorescein-5-carboxamido)hexanoic acid succinimidyl ester
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-
?
UDP-Gal + IFNalpha2b-[Tn]-FCHASE
?
show the reaction diagram
the galactosylation of human therapeutic protein IFNalpha2b[Tn] with an engineered variant of CgtB clearly demonstrates that a bacterial glycosyltransferase is able to glycosylate a human protein in vitro. FCHASE: 6-(fluorescein-5-carboxamido)hexanoic acid succinimidyl ester
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?
UDP-Gal + lyso-GM2-FCHASE
?
show the reaction diagram
FCHASE: 6-(fluorescein-5-carboxamido)hexanoic acid succinimidyl ester
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-
?
UDP-galactose + asialo-GM2
UDP + asialo-GM1
show the reaction diagram
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i.e. Gg3
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?
UDP-galactose + ganglioside GA2
UDP + ganglioside GA1
show the reaction diagram
UDP-galactose + GD2
UDP + Gd1b
show the reaction diagram
UDP-galactose + N-acetyl-D-galactosaminyl-(N-acetylneuraminyl)-D-galactosyl-1,4-beta-D-glucosyl-N-acylsphingosine
UDP + D-galactosyl-1,3-beta-N-acetyl-D-galactosaminyl-(N-acetylneuraminyl)-D-galactosyl-D-glucosyl-N-acylsphingosine
show the reaction diagram
UDPgalactose + N-acetyl-D-galactosaminyl-(N-acetylneuraminyl)-D-galactosyl-1,4-beta-D-glucosyl-N-acylsphingosine
UDP + D-galactosyl-1,3-beta-N-acetyl-D-galactosaminyl-(N-acetylneuraminyl)-D-galactosyl-D-glucosyl-N-acylsphingosine
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
UDP-galactose + asialo-GM2
UDP + asialo-GM1
show the reaction diagram
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i.e. Gg3
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?
UDP-galactose + ganglioside GA2
UDP + ganglioside GA1
show the reaction diagram
UDP-galactose + GD2
UDP + Gd1b
show the reaction diagram
UDPgalactose + N-acetyl-D-galactosaminyl-(N-acetylneuraminyl)-D-galactosyl-1,4-beta-D-glucosyl-N-acylsphingosine
UDP + D-galactosyl-1,3-beta-N-acetyl-D-galactosaminyl-(N-acetylneuraminyl)-D-galactosyl-D-glucosyl-N-acylsphingosine
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cardiolipin
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Endogenous membrane protein
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heat-stable, protease sensitive, non-dialyzable, also found in other species
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Gangliosides and ganglioside components
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most active: disialoganglioside
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N-acetylneuraminic acid
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with or without tetrahexosylceramide
N-Acetylneuraminyllactose
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weak
ovine-asialo-agalacto submaxillary mucin
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PP55B
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i.e. isopropylidene derivative of 5'-O-[[(2-decanoylamino-3-phenylpropylloxycarbonyl)amino]sulfonyl]uridine, 21% inhibition at 0.2 mM
Triton X-100
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in excess, phospholipids protect
Tween 80
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UDP-galactose
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above 0.2 mM
additional information
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no inhibition by fetuin, glucosylceramide, lactosylceramide or trihexosylceramide
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Bile salts
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activation
cardiolipin
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stimulation
Cytosolic peptide
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activation, 250fold increased reaction velocity, MW 25000, heat-labile, protease-sensitive and non-diffusible, cytosolic, MW: approximately 25 kDa, activation in a dose-dependent manner, tissue-specific
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Detergents
phosphatidylcholine
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stimulation
Triton X-100
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at room temperature 2fold increase in reation rate, in excess inhibitory
additional information
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no activation by bovine serum albumin, rat serum lipoproteins, rat liver and kidney cytosol, and phospholipids of rat cytosol and microsomes
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.68
GalNAcalpha-FCHASE
using fusion construct CgtBOH4384DELTA30-MalE (mutant with deletion of 30 amino acids from carboxy terminus + C-terminal MalE fusion)
3.18
GalNAcbeta-FCHASE
using fusion construct CgtBOH4384DELTA30-MalE (mutant with deletion of 30 amino acids from carboxy terminus + C-terminal MalE fusion)
0.01 - 0.18
N-acetyl-D-galactosaminyl-(N-acetylneuraminyl)-D-galactosyl-1,4-beta-D-glucosyl-N-acylsphingosine
1.11
UDP-Gal
using fusion construct CgtBOH4384DELTA30-MalE (mutant with deletion of 30 amino acids from carboxy terminus + C-terminal MalE fusion)
0.012 - 2.47
UDP-galactose
additional information
additional information
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kinetic properties
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.5
GalNAcalpha-FCHASE
Campylobacter jejuni
Q0P9B5, Q93MQ1, Q9K2V8
using fusion construct CgtBOH4384DELTA30-MalE (mutant with deletion of 30 amino acids from carboxy terminus + C-terminal MalE fusion)
1.07
GalNAcbeta-FCHASE
Campylobacter jejuni
Q0P9B5, Q93MQ1, Q9K2V8
using fusion construct CgtBOH4384DELTA30-MalE (mutant with deletion of 30 amino acids from carboxy terminus + C-terminal MalE fusion)
0.05
UDP-Gal
Campylobacter jejuni
Q0P9B5, Q93MQ1, Q9K2V8
using fusion construct CgtBOH4384DELTA30-MalE (mutant with deletion of 30 amino acids from carboxy terminus + C-terminal MalE fusion)
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00000005
recombinant enzyme, crude cell homogenate of transfected NIH 3T3 cells
0.000000051
recombinant enzyme, crude cell homogenate of transfected CHO-K1 cells
0.00000007
recombinant enzyme, crude cell homogenate of transfected CHO-K1 cells
0.00000009
testis, crude homogenate, native wild-type
0.463
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purified enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
assay at; assay at; assay at
6.5 - 7.2
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broad
7
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assay at
7.2 - 7.3
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adult frog brain
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
low activity content
Manually annotated by BRENDA team
expression level depends on the developmental stage
Manually annotated by BRENDA team
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oligodendrocyte cell line
Manually annotated by BRENDA team
highest content of activity
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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orientated towards the lumen
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Manually annotated by BRENDA team
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
62000
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SDS-PAGE
65000
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native PAGE, sucrose-density gradient centrifugation
additional information
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
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1 * 62000, form 1, SDS-PAGE; 1 * 65000, form 2, SDS-PAGE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
half-life at 30°C: 4 h
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, several months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
amino acid sequence determination, expression in Sf9 insect cells via baculovirus transfection; cloning and DNA sequencing, amino acid sequence determination, expression in Sf9 insect cells via baculovirus transfection; cloning and DNA sequencing, amino acid sequence determination, expression in Sf9 insect cells via baculovirus transfection; cloning and DNA sequencing, amino acid sequence determination, expression in Sf9 insect cells via baculovirus transfection
cloning and DNA sequence determination and analysis, expression in CHO-K1 and NIH-3T3 cells as fusion protein tagged with a nonapeptide from influenza virus hemagglutinin, amino acid sequence determination
cloning from human cDNA library, promotor identification via primer extension analysis, deletion and cloning of several 5'-end constructs, transient expression in human cell lines derived from oligodendroglioma and neuroblastoma LAN-5, promotor activity is cell type-specific, identification of regulatory elements
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expressed as a MalE fusion protein in Escherichia coli; expressed as a MalE fusion protein in Escherichia coli; expressed as a MalE fusion protein in Escherichia coli
expression in CHO-K1 cells as fusion protein tagged with a nonapeptide from influenza virus hemagglutinin
expression in HeLa cells, enzyme-deficient GM95 cells, CHO cells and CHOlec8 cells; expression of enzyme fragments as His-tagged peptides in Escherichia coli; in vitro transcription and translation of full length cDNA in presence of microsomes
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isolation of cDNA, expression in CHO cells, DNA and amino acid sequence determination and analysis, in vitro transcription
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stably expressed in CHO cells
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
cell culture under 42°C heat stress for 17 h, increase of gene expression to 133%
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cell culture under 42°C heat stress for 24 h, increase of gene expression to 221%
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molecular marker of breast cancer malignancy and lung metastases, significant index of tumour aggressiveness and a potential marker for the prognostic evaluation of lung metastases in breast cancer
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
DELTA20C
mutant with deletion of 20 amino acids from carboxy terminus + C-terminal MalE fusion, specific activity (mU/mg): 58.1 (substrate: GalNAcalpha), 127.5 (substrate: GalNAcbeta), 17.5 (substrate: GM2), 374 (substrate: GA2)
DELTA20N
mutant with deletion of 20 amino acids from carboxy terminus + N-terminal MalE fusion, specific activity (mU/mg): 3.3 (substrate: GalNAcalpha), 8.0 (substrate: GalNAcbeta), 2.3 (substrate: GM2), 34.4 (substrate: GA2)
DELTA30C
mutant with deletion of 30 amino acids from carboxy terminus + C-terminal MalE fusion, specific activity (mU/mg): 11.6 (substrate: GalNAcalpha), 75.7 (substrate: GalNAcbeta), 80.9 (substrate: GM2); mutant with deletion of 30 amino acids from carboxy terminus + C-terminal MalE fusion, specific activity (mU/mg): 531.5 (substrate: GalNAcalpha), 1299.5 (substrate: GalNAcbeta), 2420 (substrate: GM2), 271.5 (substrate: IFNalpha2b). kcat (1/sec): 0.5 (GalNAcalpha-FCHASE), 1.07 (GalNAcbeta-FCHASE), 0.05 (UDP-Gal), Km (mM): 1.68 (GalNAcalpha-FCHASE), 3.18 (GalNAcbeta-FCHASE), 1.11 (UDP-Gal)
DELTA30N
mutant with deletion of 30 amino acids from carboxy terminus + N-terminal MalE fusion, specific activity (mU/mg): 0.9 (substrate: GalNAcalpha), 5.3 (substrate: GalNAcbeta), 14 (substrate: GM2); mutant with deletion of 30 amino acids from carboxy terminus + N-terminal MalE fusion, specific activity (mU/mg): 49.5 (substrate: GalNAcalpha), 36 (substrate: GalNAcbeta), 96 (substrate: GM2), 14 (substrate: IFNalpha2b)
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
the galactosylation of IFNalpha2b[Tn] with an engineered variant of CgtB clearly demonstrates that a bacterial glycosyltransferase is able to glycosylate a human protein in vitro. This is the first time that the direct glycosylation of a human protein by a bacterial glycosyltransferase is reported