Information on EC 2.4.1.87 - N-acetyllactosaminide 3-alpha-galactosyltransferase

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

EC NUMBER
COMMENTARY hide
2.4.1.87
-
RECOMMENDED NAME
GeneOntology No.
N-acetyllactosaminide 3-alpha-galactosyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
UDP-alpha-D-galactose + beta-D-galactosyl-(1->4)-beta-N-acetyl-D-glucosaminyl-R = UDP + alpha-D-galactosyl-(1->3)-beta-D-galactosyl-(1->4)-beta-N-acetylglucosaminyl-R (where R can be OH, an oligosaccharide or a glycoconjugate)
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexosyl group transfer
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Glycosphingolipid biosynthesis - globo and isoglobo series
-
-
Glycosphingolipid biosynthesis - lacto and neolacto series
-
-
terminal O-glycans residues modification (via type 2 precursor disaccharide)
-
-
SYSTEMATIC NAME
IUBMB Comments
UDP-galactose:beta-D-galactosyl-(1->4)-beta-N-acetyl-D-glucosaminyl-R 3-alpha-D-galactosyltransferase
Acts on beta-galactosyl-1,4-N-acetylglucosaminyl termini on asialo-alpha1-acid glycoprotein and N-acetyllactosamine (beta-D-galactosyl-1,4-N-acetyl-beta-D-glucosamine), but not on 2'-fucosylated-N-acetyllactosamine. The non-reducing terminal N-acetyllactosamine residues of glycoproteins can also act as acceptor. Now includes EC 2.4.1.124 and EC 2.4.1.151.
CAS REGISTRY NUMBER
COMMENTARY hide
128449-51-4
-
306996-46-3
EC 2.4.1.87, formerly
78642-28-1
EC 2.4.1.124, EC 2.4.1.151, formerly
96477-57-5
EC 2.4.1.151, formerly
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
Manually annotated by BRENDA team
Alouatta sp.
new world monkey, gene GGTA1 encoding an active enzyme
-
-
Manually annotated by BRENDA team
new world monkey, gene GGTA1 encoding an active enzyme
-
-
Manually annotated by BRENDA team
old world monkeys evolved into large populations of diverse species only during the late Miocene and subsequent periods, gene GGTA1 encoding an inactivated enzyme
-
-
Manually annotated by BRENDA team
old world monkey, gene GGTA1 encoding an inactivated enzyme
-
-
Manually annotated by BRENDA team
old world mokey, gene GGTA1 encoding an inactivated enzyme
-
-
Manually annotated by BRENDA team
gene GGTA1 encoding an inactivated enzyme
-
-
Manually annotated by BRENDA team
apes, gene GGTA1 encoding an inactivated enzyme
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
Lemuroidea
gene GGTA1 encoding an active enzyme
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
old world monkey, gene GGTA1 encoding an inactivated enzyme
-
-
Manually annotated by BRENDA team
gene GGTA1 encoding an active enzyme
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
no activity in african green monkey
-
-
-
Manually annotated by BRENDA team
nonprimate mammals
gene GGTA1 encoding an active enzyme
-
-
Manually annotated by BRENDA team
gene GGTA1 encoding an inactivated enzyme
-
-
Manually annotated by BRENDA team
new world monkeys, gene GGTA1 encoding an active enzyme
-
-
Manually annotated by BRENDA team
Sumatra, gene GGTA1 encoding an inactivated enzyme
-
-
Manually annotated by BRENDA team
Borneo, gene GGTA1 encoding an inactivated enzyme
-
-
Manually annotated by BRENDA team
new world monkey, gene GGTA1 encoding an active enzyme
-
-
Manually annotated by BRENDA team
serotype 6B
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
-
absence of galactose-alpha-1,3-galactose residue in the GalT-KO pig cells results in a higher level of overall fucosylated N-glycans in GalT-KO pig fibroblasts compared to wild-type pig fibroblasts, the relative quantity of the N-glycolylneuraminic acid antigen is also slightly higher in the GalT-KO pigs, mutant phenotype, overview
metabolism
physiological function
-
the wciN gene encodes a galactosyltransferase involved in the repeating unit assembly of Pneumococcus type 6B capsular polysaccharide
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
UDP-2-acetonyl-2-deoxy-galactose + asialofetuin
UDP + ?
show the reaction diagram
-
mutant enzymes SGG and AGG, 37C, MnCl2, 25 mM Tris-HCl, pH 7.0
-
-
?
UDP-2-acetonyl-2-deoxy-galactose + N-acetyllactosaminyl-chitotriose
UDP + ?
show the reaction diagram
-
mutant enzymes SGG, AGG, and TGG, 37C, MnCl2, 25 mM Tris-HCl, pH 7.0
-
-
?
UDP-alpha-D-galactose + beta-D-galactosyl-(1->4)-beta-N-acetyl-D-glucosaminyl-R
UDP + alpha-D-galactosyl-(1->3)-beta-D-galactosyl-(1->4)-beta-N-acetylglucosaminyl-R
show the reaction diagram
UDP-alpha-D-galactose + Glcalpha-diphospho-O(CH2)10CH3
UDP + Galalpha(1->3)Glcalpha-diphospho-O(CH2)10CH3
show the reaction diagram
-
wciN encodes an alpha-1,3-galactosyltransferase catalyzing the transfer of galactosyl from UDP-Gal onto the Glcalpha-diphosphate-lipid acceptor to form Galalpha(1->3)Glcalpha-diphosphate-lipid
-
-
?
UDP-alpha-D-galactose + N-acetyllactosamine
UDP + ?
show the reaction diagram
-
-
-
-
?
UDP-Gal + Gal-beta-1,4-GlcNAc-(CH2)8CO2CH3
UDP + ?
show the reaction diagram
-
-
-
-
?
UDP-galactose + alpha-L-Fucp-1,2-beta-D-Galp-O(CH2)7CH3
UDP + ?
show the reaction diagram
-
The presence of UDP and Mn2+ has a marked influence on the thermodynamic parameters enthalpy and entropy of association, they decrease, binding to substrate is independent of UDP at physiological temperatures, but cooperatively is observed at lower temperatures
-
-
?
UDP-galactose + asialo-alpha1-acid glycoprotein
UDP + alpha-D-galactosyl-asialo-alpha1-acid glycoprotein
show the reaction diagram
-
-
-
-
?
UDP-galactose + asialofetuin
UDP + alpha-D-galactosylasialofetuin
show the reaction diagram
-
-
-
-
?
UDP-galactose + beta-D-Gal(1-3)beta-D-GlcNAc(1-3)beta-D-Gal(1-4)-D-Glc
UDP + alpha-D-Gal(1-3)beta-D-Gal(1-3)beta-D-GlcNAc(1-3)beta-D-Gal(1-4)-D-Glc
show the reaction diagram
-
-
-
-
?
UDP-galactose + beta-D-Gal(1-4)-D-GlcNAc(1-2)-[beta-D-Gal(1-4)beta-D-GlcNAc(1-6)]-D-Man
?
show the reaction diagram
-
-
-
-
?
UDP-galactose + beta-D-Gal(1-4)beta-D-GlcNAc(1-2)-D-Man
UDP + alpha-D-Gal(1-3)beta-D-Gal(1-4)beta-D-GlcNAc(1-2)-D-Man
show the reaction diagram
-
-
-
-
?
UDP-galactose + beta-D-Gal(1-4)beta-D-GlcNAc(1-6)-D-Man
UDP + alpha-D-Gal(1-3)beta-D-Gal(1-4)beta-D-GlcNAc(1-6)-D-Man
show the reaction diagram
-
-
-
-
?
UDP-galactose + beta-D-Gal(1-4)GlcNAcbeta(1-2)Manalpha(1-3)Manbeta(1-4)GlcNAc
UDP + alpha-D-Gal(1-3)beta-D-Gal(1-4)GlcNAcbeta(1-2)Manalpha(1-3)Manbeta(1-4)GlcNAc
show the reaction diagram
-
-
-
-
?
UDP-galactose + beta-D-galactosyl-1,4-D-glucose
UDP + alpha-D-galactosyl-1,3-beta-D-galactosyl-1,4-D-glucose
show the reaction diagram
-
-
-
-
?
UDP-galactose + beta-D-galactosyl-1,4-N-acetyl-D-glucosamine
UDP + alpha-D-galactosyl-1,3-beta-D-galactosyl-1,4-N-acetyl-D-glucosamine
show the reaction diagram
UDP-galactose + beta-D-galactosyl-1,4-N-acetyl-D-glucosaminyl-R
UDP + alpha-D-galactosyl-1,3-beta-D-galactosyl-1,4-N-acetyl-D-glucosaminyl-R
show the reaction diagram
UDP-galactose + D-galactose
UDP + alpha-D-galactosyl-1,3-D-galactoside
show the reaction diagram
-
-
-
-
?
UDP-galactose + galactosyl-1,3-N-acetylglucosaminyl-1,3-galactosyl-1,4-glucosyl-1,1-ceramide
UDP + O-alpha-galactosyl-1,3-galactosyl-1,3-N-acetylglucosaminyl-1,3-galactosyl-1,4-glucosyl-1,1-ceramide
show the reaction diagram
-
biosynthesis of blood group B specific pentaglycosylceramide
-
-
?
UDP-galactose + galactosyl-1,3-N-acetylglucosaminyl-1,3-galactosyl-1,4-glucosyl-1,1-ceramide
UDP + O-alpha-galactosyl-1,3-galactosyl-1,3-N-acetylglucosaminyl-1,3-galactosyl-1,4-glucosyl-1,1-ceramide + O-alpha-galactosyl-1,4-galactosyl-1,3-N-acetylglucosaminyl-1,3-galactosyl-1,4-glucosyl-1,1-ceramide
show the reaction diagram
-
specific for UDP-galactose as donor substrate
-
?
UDP-galactose + galactosyl-1,3-N-acetylglucosaminyl-1,3-galactosyl-1,4-glucosyl-1,1-ceramide
UDP + O-alpha-galactosyl-1,4-galactosyl-1,3-N-acetylglucosaminyl-1,3-galactosyl-1,4-glucosyl-1,1-ceramide
show the reaction diagram
-
biosynthesis of blood group B specific pentaglycosylceramide
-
?
UDP-galactose + Galbeta(1-4)GlcNAcbeta(1-3)(Galbeta(1-4)GlcNAcbeta(1-6))Galbeta(1-4)GlcNAcbeta(1-3)[Galbeta(1-4)GlcNAcbeta(1-3)(Galbeta(1-4)GlcNAc(1-6)Galbeta(1-4)GlcNAcbeta(1-6))]Galbeta(1-4)GlcNAc
UDP + Galalpha(1-3)Galbeta(1-4)GlcNAcbeta(1-3)(Galalpha(1-3)Galbeta(1-4)GlcNAcbeta(1-6))Galbeta(1-4)GlcNAcbeta(1-3)[Galalpha(1-3)Galbeta(1-4)GlcNAcbeta(1-3)(Galalpha(1-3)Galbeta(1-4)GlcNAc(1-6))Galbeta(1-4)GlcNAcbeta(1-6)]Galbeta(1-4)GlcNAc
show the reaction diagram
-
-
-
?
UDP-galactose + glycoprotein
UDP + alpha-D-galactosylglycoprotein
show the reaction diagram
UDP-galactose + H2O
UDP + D-galactose
show the reaction diagram
-
-
-
?
UDP-galactose + lactose
UDP + ?
show the reaction diagram
UDP-galactose + lactose
UDP + alpha-D-galactosyl-(1-3)-beta-D-galactosyl-(1-4)-beta-D-glucose
show the reaction diagram
UDP-galactose + lactose
UDP + alpha-D-galactosyl-(1->3)-beta-D-galactosyl-(1->4)-D-glucose
show the reaction diagram
UDP-galactose + Manalpha(1->2)Manalpha(1->2)Man-pyridylamine
UDP + Galalpha(1->3)Manalpha(1->2)Manalpha(1->2)Man-pyridylamine
show the reaction diagram
UDP-galactose + N-acetyllactosamine
UDP + ?
show the reaction diagram
-
-
-
-
?
UDP-galactose + N-acetyllactosamine
UDP + alpha-D-galactosyl-1,3-beta-D-galactosyl-1,4-N-acetyl-D-glucosamine
show the reaction diagram
UDP-galactose + p-nitrophenyl-alpha-D-galactoside
UDP + ?
show the reaction diagram
-
-
-
-
?
UDP-galactose + p-nitrophenyl-beta-D-galactoside
UDP + ?
show the reaction diagram
-
-
-
-
?
UDP-galactose + saccharides containing Galalpha(1-3)Gal epitope
?
show the reaction diagram
AF415202
-
-
-
?
UDP-galactose + substituted LacNAc-saccharides containing Galalpha(1-3)Gal epitope
?
show the reaction diagram
UDP-N-acetyl-azido-galactosamine + N-acetyllactosaminyl-chitotriose
UDP + ?
show the reaction diagram
-
mutant enzymes SGG, AGG, and TGG, 37C, MnCl2, 25 mM Tris-HCl, pH 7.0
-
-
?
UDP-N-acetylgalactosamine + lactose
UDP + ?
show the reaction diagram
UDP-N-acetylgalactosamine + N-acetyllactosamine
UDP + ?
show the reaction diagram
-
no reaction of wild-type, only of AGGL mutant
-
-
?
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
UDP-alpha-D-galactose + beta-D-galactosyl-(1->4)-beta-N-acetyl-D-glucosaminyl-R
UDP + alpha-D-galactosyl-(1->3)-beta-D-galactosyl-(1->4)-beta-N-acetylglucosaminyl-R
show the reaction diagram
UDP-alpha-D-galactose + N-acetyllactosamine
UDP + ?
show the reaction diagram
-
-
-
-
?
UDP-galactose + alpha-L-Fucp-1,2-beta-D-Galp-O(CH2)7CH3
UDP + ?
show the reaction diagram
-
The presence of UDP and Mn2+ has a marked influence on the thermodynamic parameters enthalpy and entropy of association, they decrease, binding to substrate is independent of UDP at physiological temperatures, but cooperatively is observed at lower temperatures
-
-
?
UDP-galactose + beta-D-galactosyl-1,4-N-acetyl-D-glucosaminyl-R
UDP + alpha-D-galactosyl-1,3-beta-D-galactosyl-1,4-N-acetyl-D-glucosaminyl-R
show the reaction diagram
UDP-galactose + galactosyl-1,3-N-acetylglucosaminyl-1,3-galactosyl-1,4-glucosyl-1,1-ceramide
UDP + O-alpha-galactosyl-1,3-galactosyl-1,3-N-acetylglucosaminyl-1,3-galactosyl-1,4-glucosyl-1,1-ceramide
show the reaction diagram
-
biosynthesis of blood group B specific pentaglycosylceramide
-
-
?
UDP-galactose + galactosyl-1,3-N-acetylglucosaminyl-1,3-galactosyl-1,4-glucosyl-1,1-ceramide
UDP + O-alpha-galactosyl-1,4-galactosyl-1,3-N-acetylglucosaminyl-1,3-galactosyl-1,4-glucosyl-1,1-ceramide
show the reaction diagram
-
biosynthesis of blood group B specific pentaglycosylceramide
-
?
UDP-galactose + lactose
UDP + ?
show the reaction diagram
-
wild-type enzyme, 37C, MnCl2, 25 mM Tris-HCl, pH 7.0
-
-
?
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
-
slight activation
Zn2+
-
slight activation
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
alpha-lactalbumin
-
-
-
diethyl dicarbonate
-
-
Galbeta(1-3)GlcNAc-C8
-
strong
-
octyl 3-amino-3-deoxy-3-N-(2-diazo-3,3,3-trifluoropropionyl-beta-D-galactopyranosyl-(1,4)-2-acetamido-2-deoxy-beta-D-glucopyranoside)
-
i.e. DTFP-LacNAc-C8
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Triton X-100
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.25 - 3.7
asialo-alpha1-acid glycoprotein
-
16
beta-D-Gal(1-3)beta-D-GlcNAc(1-3)beta-D-Gal(1-4)-D-Glc
-
-
9
beta-D-Gal(1-4)-D-Glc
-
-
1.15 - 1.39
beta-D-Gal(1-4)-D-GlcNAc
0.1
beta-D-Gal(1-4)-D-GlcNAc(1-2)-[beta-D-Gal(1-4)beta-D-GlcNAc(1-6)]-D-Man
-
-
0.25
beta-D-Gal(1-4)beta-D-GlcNAc(1-2)-D-Man
-
-
0.31
beta-D-Gal(1-4)beta-D-GlcNAc(1-6)-D-Man
-
-
392
D-galactose
-
10 mM MnCl2, 0.3 mM UDP-galactose
0.31
Gal-beta-1,4-GlcNAc-(CH2)8CO2CH3
-
-
1.67
galactosyl-1,3-N-acetylglucosaminyl-1,3-galactosyl-1,4-glucosyl-1,1-ceramide
-
-
0.57
Galbeta(1-4)GlcNAcbeta(1-2)Man(1-3)Manbeta(1-4)GlcNAc
-
-
4.5 - 330
lactose
0.6 - 15
N-acetyllactosamine
107
p-nitrophenyl-alpha-D-galactoside
-
10 mM MnCl2, 0.3 mM UDP-galactose
1
p-nitrophenyl-beta-D-galactoside
-
10 mM MnCl2, 0.3 mM UDP-galactose
0.67
UDP-Gal
-
-
0.00069 - 270
UDP-galactose
0.068 - 45.8
UDP-N-acetylgalactosamine
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.32
D-galactose
Bos taurus
-
10 mM MnCl2, 0.3 mM UDP-galactose
0.14 - 6.4
lactose
1.9
N-acetyllactosamine
Bos taurus
-
10 mM MnCl2, 0.3 mM UDP-galactose
0.059
p-nitrophenyl-alpha-D-galactoside
Bos taurus
-
10 mM MnCl2, 0.3 mM UDP-galactose
1.1
p-nitrophenyl-beta-D-galactoside
Bos taurus
-
10 mM MnCl2, 0.3 mM UDP-galactose
0.00013 - 6.4
UDP-galactose
0.045 - 0.4
UDP-N-acetylgalactosamine
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0008
D-galactose
Bos taurus
-
10 mM MnCl2, 0.3 mM UDP-galactose
71
0.000074 - 0.75
lactose
114
3.2
N-acetyllactosamine
Bos taurus
-
10 mM MnCl2, 0.3 mM UDP-galactose
1116
0.0006
p-nitrophenyl-alpha-D-galactoside
Bos taurus
-
10 mM MnCl2, 0.3 mM UDP-galactose
5591
1.1
p-nitrophenyl-beta-D-galactoside
Bos taurus
-
10 mM MnCl2, 0.3 mM UDP-galactose
1624
0.5 - 4.3
UDP-galactose
129
0.5 - 0.6
UDP-N-acetylgalactosamine
1308
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.27
Galbeta(1-3)GlcNAc-C8
-
-
-
0.3
octyl 3-amino-3-deoxy-3-N-(2-diazo-3,3,3-trifluoropropionyl-beta-D-galactopyranosyl-(1,4)-2-acetamido-2-deoxy-beta-D-glucopyranoside)
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.001
-
membrane fraction
1.1
-
wild-type, UDP-galactose as donor, lactose as acceptor, 37C, MnCl2, 25 mM Tris-HCl, pH 7.0
4.3
-
-
265
-
purified enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 7
-
cacodylate buffer
6.1
-
assay at
7
-
assay at
8
-
assay at
additional information
-
charge heterogenity, pI varying between 5.0 and 6.5
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.3 - 7
-
pH 5.3: about 45% of activity maximum, pH 7.0: about 30% of activity maximum
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
34483
-
2 * 34483
38799
-
x * 38799, calculated from amino acid sequence
39000
-
x * 39000, SDS-PAGE
43000
-
gel filtration
80000
-
x * 80000, SDS-PAGE
additional information
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
-
2 * 34483
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
5% PEG 6000, 0.1 M Tris-HCl, pH 8.0, 10 mM MnCl2, 10 mM UDP, in complex with substrate p-nitrophenyl-beta-galactoside, 25% glycerol as cryoprotectant
-
AGGL-mutant in complex with UDP-N-acetylgalactosamine with 20 mM MES-NaOH buffer, pH 6, 10% glycerol, 10 mM MnCl2, 10 mM UDP-N-acetylgalactosamine and reservoir solution (10-15% PEG 6000, 0.1 M Tris-HCl, pH 8, 15-25% MPD), and RAAI-mutant with 0.1 M Tris-HCl, pH 8, 10-50% PEG 4000, 0.2 M sodium acetate, 10 mM MnCl2, 10 mM UDP-galactose with the same reservoir solution, vapor diffusion hanging drop method
-
apo crystals of alpha3GT are grown by the vapour-diffusion, hanging-drop method. Structure of a complex containing an inhibitory analogue of UDP-galactose, UDP-2F-galactose, in a complex with the Arg365Lys mutant of alpha3GT. The binding of a donor substrate analogue induces conformational changes in both the ligand and the enzyme. Two loops of alpha3GT are stabilized in the complex of which the C-terminal region, in particular, is highly flexible. Structural transitions in this region are connected with donor substrate binding and distortion (ground state destabilization), cleavage of the UDP to galactose bond, formation of a binding site for acceptor substrate and UDP release
crystal structure of the Glu317Gln mutant in complex with UDP-Gal is reported; purified recombinant enzyme mutants with bound UDP-Gal and Mn2+, hanging drop vapour diffusion at 16C, mixing of 0.001 ml protein solution containing 5 mg/ml mutant E317Q in 20 mM MES-NaOH buffer, pH 6.0, and 10% glycerol, containing 10 mM MnCl2 and 10 mM UDP-Gal with 0.001 ml of reservoir solution containing 10% PEG 6000, 0.1 M Tris-HCl, pH 8.0, and 8% MPD, for mutant D316N and D316E, 0.001 ml of 5 mg/ml protein in in 20 mM MES-NaOH buffer, pH 6.0, with 10% glycerol, 10 mM UDP-Gal, 10 mM MnCl2, and 10 mM N-acetyllactosamine, are mixed with 0.001 ml of a reservoir solution containing 5-10% PEG 6000, 0.1 M Tris-HCl, pH 8.0, and 5-14% MPD, X-ray diffraction structure determination and analysis at 1.77-2.2 A resolution
-
structure analysis of the enzyme in ternary complex with substrate UDP-2F-Gal, crystal structure fold-type A, PDB ID 2VFZ
-
vapor diffusion hanging drop method
-
vapour diffusion hanging drop method
-
structure analysis of the enzyme in ternary complex with substrates UDP and lactose, crystal structure fold-type A, PDB ID 1GWF
-
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
detergent: rapid and irreversible inactivation in absence of detergent, more than 50% loss of activity after 4-6 weeks
-
mutants are not stable under assay conditions (10 mM Tris-HCl, pH 7.4, 37C), but for 20 min with 10 mM MES, pH 6.0, at 30C
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 20 mM MES, pH 6.2, 50 mM NaCl, detergent, glycerol, stable for several months
-
0C, more than 50% loss of activity after 4-6 weeks
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
centrifugation of cell culture, pellet lysed with EasyLyse, loaded onto Ni-chelate column, washing and elution with 20 mM Tris-HCl, pH 7.9, containing NaCl and imidazole
-
different detergents for solubilization, best with Triton CF-54; partial
-
Ehrlich ascites carcinoma cells
-
Ehrlich ascites carcinoma cells; partial
-
HisTrap HP column chromatography
-
partial
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a single gene locus specifies 4 isoforms; characterization of cDNA
-
catalytic domain (residues 80-386) is expressed in Escherichia coli
-
cloned from cDNA library, expression of full-length and truncated DNA in Escherichia coli and COS-1 cells, the truncated sequences are fused to protein A sequence resulting in expression of soluble chimeric proteins, DNA sequence determination and analysis
-
cloned from genomic and cDNA library and macrophages, DNA sequence analysis, alternative splicing
cloning of C-terminal domain
expressed in Escherichia coli and COS-7 cells as a His-tagged fusion protein
-
expressed in Escherichia coli BL21(DE3) cells
-
expression in COS-7 cells
-
expression in COS-7 cells, promotor sequences A-C determination and analysis, gene regulation and regulatory sequences, alternative splicing
AF415202
expression in Escherichia coli
-
gene GGTA1, partial sequence comparisons and phylogenetic analysis
murine alpha(1,3)GT gene is cloned into the Moloney murine leukemia retroviral vector, pLNCX, giving rise to pLNCKG expression in A549 human lung tumor cells
-
mutants W249G, W250Y, W250F, W315Y anf W356T, of catalytic domain are constructed and expressed in Escherichia coli BL21
-
PCR megaprimer method, expressed in Escherichia coli BL21(DE3)
-
PCR-amplification, expression in Escherichia coli BL21(DE3) LysS
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D316E
-
mutant show modest reduction in kcat and Km for lactose. Strucutural studies with mutant D316E show that the negative charge is crucial for catalytic activity and needed for its interaction with Arg202 for an active site structure that facilitates the binding of UDP-gal in a catalytically competent conformation; site-directed mutagenesis, a catalytic domain mutant, crystal structure determination with bound UDP-Gal and Mn2+
D316N
-
mutant is inactive. Strucutural studies with mutant D316N show that the negative charge is crucial for catalytic activity and needed for its interaction with Arg202 for an active site structure that facilitates the binding of UDP-gal in a catalytically competent conformation; site-directed mutagenesis, a catalytic domain mutant, crystal structure determination with bound UDP-Gal and Mn2+
E317A
-
relative activity of mutant protein: 0.1% (compared to wild-type 100%)
E317A/E317Q
-
site-directed mutagenesis, substrate binding compared to wild-type
E317C
-
relative activity of mutant protein: 0.8% (compared to wild-type 100%)
E317D
-
relative activity of mutant protein: 0.4% (compared to wild-type 100%)
E317H
-
relative activity of mutant protein: 0.1% (compared to wild-type 100%)
H280A/A281A/A282A
-
mutant AAA, 20 or 200 mM lactose as acceptor, 14 and 41% galactosyltransferase activity (UDP-galactose as donor), 4 and 6% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37C, MnCl2, 25 mM Tris-HCl, pH 7.0
H280A/A281G/A282G
-
mutant AGG, 20 or 200 mM lactose as acceptor, 0.4 and 1% galactosyltransferase activity (UDP-galactose as donor), 0.3 and 2% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37C, MnCl2, 25 mM Tris-HCl, pH 7.0, among the mutants with highest N-acetylgalactosamnine transferase activity and ability to transfer C2-modified sugars
H280A/A281G/A282G/L283I
-
AGGI-mutant, instable, leucin 283 is important for enzyme stability
H280A/A281G/A282G/L283L
-
AGGL-mutant, higher N-acetylgalactosamine transferase activity than galactosyltransferase activity, leucin 283 is important for enzyme stability
H280A/A281G/A282G/L283V
-
AGGV-mutant, instable, leucin 283 is important for enzyme stability
H280G/A281A/A282A
-
protein unstable, precipitating during purification
H280G/A281A/A282G
-
mutant GAG, 20 or 200 mM lactose as acceptor, 5 and 27% galactosyltransferase activity (UDP-galactose as donor), 4 and 6% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37C, MnCl2, 25 mM Tris-HCl, pH 7.0
H280G/A281G/A282G/L283I
-
GGGI-mutant, instable, leucin 283 is important for enzyme stability
H280G/A281G/A282G/L283L
-
GGGL-mutant, higher N-acetylgalactosamine transferase activity than galactosyltransferase activity, leucin 283 is important for enzyme stability
H280G/A281G/A282G/L283V
-
GGGV-mutant, instable, leucin 283 is important for enzyme stability
H280L/A281G/A282G
-
mutant LGG, 20 or 200 mM lactose as acceptor, 2 and 1% galactosyltransferase activity (UDP-galactose as donor), 3 and 2% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37C, MnCl2, 25 mM Tris-HCl, pH 7.0
H280Q
-
80fold reduction in catalytic activity
H280R/A281A/A282A/L283I
-
RAAI-mutant, no catalytic activity, crystallisation
H280S/A281G/A282G
-
mutant SGG, 20 or 200 mM lactose as acceptor, 5 and 5% galactosyltransferase activity (UDP-galactose as donor), 2 and 11% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37C, MnCl2, 25 mM Tris-HCl, pH 7.0, among the mutants with highest GalNac transferase activity and ability to transfer C2-modified sugars
H280T/A281G/A282G
-
mutant TGG, 20 or 200 mM lactose as acceptor, 3 and 15% galactosyltransferase activity (UDP-galactose as donor), 1 and 6% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37C, MnCl2, 25 mM Tris-HCl, pH 7.0
H280V/A281G/A282G
-
mutant VGG, 20 or 200 mM lactose as acceptor, 4 and 16% galactosyltransferase activity (UDP-galactose as donor), 0.5 and 3% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37C, MnCl2, 25 mM Tris-HCl, pH 7.0
H315Q
-
mutant protein shows modest changes in kinetic parameters for lactose
H315R
-
mutant shows a major losss in catalytic activity arising from a 500fold reduction in kcat
H319A
-
mutant shows a 2fold reduction for kcat
H319E
-
mutant is inactive
H319Y
-
mutant is inactive
Q247E
-
50fold reduction in turnover number for transferase reaction, unchanged hydrolase activity, Km-values for UDP-galactose and lactose are similar to the wild-type values
R365K
-
mutation reduces turnover-number for UDP-galactose hydrolysis about 10fold but has no significant effect on the affinity for UDP-galactose
S318A
-
mutant shows a 10fold reduction for kcat
W249G
-
1.95fold increase in Km-value for UDP-galactose in galactosyltransferase reaction, turnover number of hydrolase reaction is identical to wild-type value, 2.1fold increase in Km-value for UDP-galactose in hydrolase reaction. Mutation does not affect the overall structure of the enzyme or its interactions with ligands
W250F
-
2.6fold increase in turnover number of galactosyltransferase reaction, 9.3fold increase in Km-value for UDP-galactose in galactosyltransferase reaction, 1.1fold decrease in Km-value for lactose, 1.4fold increase in turnover number of hydrolase reaction, 1.5fold increase in Km-value for UDP-galactose in hydrolase reaction
W250Y
-
1.2fold increase in turnover number of galactosyltransferase reaction, 5.2fold increase in Km-value for UDP-galactose in galactosyltransferase reaction, 2fold decrease in Km-value for lactose, 1.8fold increase in turnover number of hydrolase reaction, 2.3fold increase in Km-value for UDP-galactose in hydrolase reaction
W314Y
-
29fold decrease in turnover number of galactosyltransferase reaction, 1.67fold increase in Km-value for UDP-galactose in galactosyltransferase reaction, 2fold decrease in Km-value for lactose, 1.1fold increase in turnover number of hydrolase reaction, 1.4fold increase in Km-value for UDP-galactose in hydrolase reaction. Mutation does not affect the overall structure of the enzyme or its interactions with ligands
W356T
-
12.3fold decrease in turnover number of galactosyltransferase reaction, 2fold increase in Km-value for UDP-galactose in galactosyltransferase reaction, 6.3fold increase in Km-value for lactose, 14.5fold decrease in turnover number of hydrolase reaction, 2.5fold increase in Km-value for UDP-galactose in hydrolase reaction
N375V
-
exchange and truncation of terminal V376, 70% loss of activity
D102A
-
inactive
D104A
-
inactive
D116A
-
the mutant remains active
D118A
-
the mutant remains active
D28A
-
the mutant remains active
D30A
-
the mutant remains active
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
diagnostics
-
identification of N-acetyllactosamine moiety of glycoproteins and glycolipids by chemiluminescence with mutated enzymes
medicine
synthesis