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Information on EC 2.4.1.135 - galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase and Organism(s) Homo sapiens and UniProt Accession Q9P2W7

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IUBMB Comments
Involved in the biosynthesis of the linkage region of glycosaminoglycan chains as part of proteoglycan biosynthesis (chondroitin, dermatan and heparan sulfates). Requires Mn2+.
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Homo sapiens
UNIPROT: Q9P2W7
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The taxonomic range for the selected organisms is: Homo sapiens
The enzyme appears in selected viruses and cellular organisms
Synonyms
glcat-i, glcat-p, glcat-1, glucuronosyltransferase i, beta1,3-glucuronosyltransferase i, glucuronyltransferase-i, glcuat-i, glcati, beta-1,3-glucuronyltransferase 1, beta1,3-glucuronosyltransferase-i, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
beta1,3-glucuronosyltransferase I
-
glycosaminoglycan glucuronyltransferase I
-
beta1,3-glucuronosyltransferase I
-
-
beta1,3-glucuronyltransferase I
-
-
-
-
Galbeta1,3-glucuronosyltransferase
-
-
-
-
GlcAT-I
glucuronosyltransferase I
-
-
-
-
glucuronyltransferase-I
-
-
glycosyltransferases GlcAT1
-
-
UDP-GlcUA:Gal Beta-1,3-Gal-R glucuronyltransferase
-
-
-
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UDP-GlcUA:glycoprotein beta-1,3-glucuronyltransferase
-
-
-
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UDP-glucuronic acid:Galbeta1,3Gal-R glucuronsyltransferase
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexosyl group transfer
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
UDP-alpha-D-glucuronate:[protein]-3-O-(beta-D-galactosyl-(1->3)-beta-D-galactosyl-(1->4)-beta-D-xylosyl)-L-serine D-glucuronosyltransferase (configuration-inverting)
Involved in the biosynthesis of the linkage region of glycosaminoglycan chains as part of proteoglycan biosynthesis (chondroitin, dermatan and heparan sulfates). Requires Mn2+.
CAS REGISTRY NUMBER
COMMENTARY hide
227184-75-0
-
9030-08-4
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
UDP-Gal + Galbeta(1-3)Gal
Galbeta(1-3)Galbeta(1-3)Gal + UDP
show the reaction diagram
no activity with wild-type enzyme, weak activity with mutant enzyme H308R
-
-
?
UDP-galacturonic acid + Galbeta(1-3)Gal
Galbeta(1-3)Galbeta(1-3)Gal + UDP
show the reaction diagram
-
-
-
?
UDP-GlcNAc + Galbeta(1-3)Gal
GlcNAcbeta(1-3)Galbeta(1-3)Gal + UDP
show the reaction diagram
no activity with wild-type enzyme, activity with mutant enzyme H308R is nearly equal to activity with UDP-Glc
-
-
?
UDP-glucose + galactosyl-1,3-thiogalactose
?
show the reaction diagram
-
-
-
?
UDP-glucuronate + Galbeta(1-3)Gal
GlcAbeta(1-3)Galbeta(1-3)Gal + UDP
show the reaction diagram
-
-
-
?
UDP-Man + Galbeta(1-3)Gal
Manbeta(1-3)Galbeta(1-3)Gal + UDP
show the reaction diagram
no activity with wild-type enzyme, efficient reaction with mutant enzyme H308R
-
-
?
Galbeta(1-3)Gal(6-O-sulfate)beta(1-4)Xyl(2-O-phosphate)beta1-O-Ser + UDP-GlcUA
GlcUAbeta(1-3)Galbeta(1-3)Gal(6-O-sulfate)beta(1-4)Xyl(2-O-phosphate)beta1-O-Ser + UDP
show the reaction diagram
-
-
-
-
?
Galbeta(1-3)Gal(6-O-sulfate)beta(1-4)Xylbeta1-O-Ser + UDP-GlcUA
GlcUAbeta(1-3)Galbeta(1-3)Gal(6-O-sulfate)beta(1-4)Xylbeta1-O-Ser + UDP
show the reaction diagram
-
-
-
-
?
Galbeta(1-3)Galbeta(1-4)Xyl(2-O-phosphate)beta1-O-Ser + UDP-GlcUA
GlcUAbeta(1-3)Galbeta(1-3)Galbeta(1-4)Xyl(2-O-phosphate)beta1-O-Ser + UDP
show the reaction diagram
-
-
-
-
?
Galbeta(1-3)Galbeta(1-4)Xylbeta1-O-Ser + UDP-GlcUA
GlcUAbeta(1-3)Galbeta(1-3)Galbeta(1-4)Xylbeta1-O-Ser + UDP
show the reaction diagram
-
-
-
-
?
UDP-glucuronate + 3-beta-D-galactosyl-4-beta-D-galactosyl-O-beta-D-xylosylprotein
UDP + 3-beta-D-glucuronosyl-3-beta-D-galactosyl-4-beta-D-galactosyl-O-beta-D-xylosylprotein
show the reaction diagram
-
plays a key role in glycosaminoglycan biosynthesis
-
-
?
UDP-glucuronate + Galbeta(1-3)Gal(6-sulfate)beta1-O-methoxyphenyl
UDP + ?
show the reaction diagram
-
-
-
?
UDP-glucuronate + Galbeta(1-3)Galbeta(1-4)Xyl
UDP + GlcAbeta(1-3)-Galbeta(1-3)Galbeta(1-4)Xyl
show the reaction diagram
UDP-glucuronate + Galbeta(1-3)Galbeta1-O-methoxyphenyl
UDP + ?
show the reaction diagram
-
-
-
?
UDP-glucuronate + Galbeta1-3Gal
UDP + Galbeta1-3Galbeta1-3Gal
show the reaction diagram
-
the enzyme exhibits a strict selectivity towards Galbeta1-3Gal structures
-
-
?
additional information
?
-
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
UDP-glucuronate + 3-beta-D-galactosyl-4-beta-D-galactosyl-O-beta-D-xylosylprotein
UDP + 3-beta-D-glucuronosyl-3-beta-D-galactosyl-4-beta-D-galactosyl-O-beta-D-xylosylprotein
show the reaction diagram
-
plays a key role in glycosaminoglycan biosynthesis
-
-
?
UDP-glucuronate + Galbeta(1-3)Galbeta(1-4)Xyl
UDP + GlcAbeta(1-3)-Galbeta(1-3)Galbeta(1-4)Xyl
show the reaction diagram
-
high expression of the GlcAT-I gene renders the cells capable of synthesizing the HNK-1 epitope
-
-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
stimulation at 10 mM is 25% of the stimulation with Mn2+. No further activation when the concentration is increased up to 50 mM
Mg2+
stimulation at 10 mM is 50% of the stimulation with Mn2+. No further activation when the concentration is increased up to 50 mM
Mn2+
0.1-10 mM strongly inactivates. No further activation when the concentration is increased up to 50 mM. Not only essential for GlcAT-I activity but also required for cosubstrate binding. The Asp194-Asp195-Asp196 motif is a major element of the UDP/Mn2+ binding site
Zn2+
stimulation at 10 mM is 50% of the stimulation with Mn2+. No further activation when the concentration is increased up to 50 mM
Cu2+
-
divalent cations essential, 2.7% as effective as Mn2+
Mg2+
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divalent cations essential, 5.5% as effective as Mn2+
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,3-Butanedione
irreversible. UDP-glucuronic acid protects, no protection by UDP-glucose. Activity of H308R towards UDP-glucose is unaffected by the reagent
N-Phenylmaleimide
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0499 - 0.059
UDP-GlcA
0.108
UDP-GlcNAc
pH 5.0, 37°C, mutant enzyme H308R
0.233
UDP-glucose
pH 5.0, 37°C, mutant enzyme H308R
0.074
UDP-Man
pH 5.0, 37°C, mutant enzyme H308R
0.035
Galbeta(1-3)Gal(6-O-sulfate)beta(1-4)Xyl(2-O-phosphate)beta1-O-Ser
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pH 6.5, 2 mM MnCl2, 37°C
0.049
Galbeta(1-3)Gal(6-O-sulfate)beta(1-4)Xylbeta1-O-Ser
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pH 6.5, 2 mM MnCl2, 37°C
0.0804
Galbeta(1-3)Galbeta(1-4)Xyl
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-
0.025
Galbeta(1-3)Galbeta(1-4)Xyl(2-O-phosphate)beta1-O-Ser
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pH 6.5, 2 mM MnCl2, 37°C
0.046
Galbeta(1-3)Galbeta(1-4)Xylbeta1-O-Ser
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pH 6.5, 2 mM MnCl2, 37°C
4.48
Galbeta1-3Gal
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37°C, pH 6.5
0.04 - 1.27
UDP-glucuronate
0.0293 - 0.287
UDPglucuronate
additional information
additional information
-
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
Manually annotated by BRENDA team
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
B3GA1_HUMAN
334
1
38256
Swiss-Prot
Secretory Pathway (Reliability: 1)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
43000
-
2 * 43000, SDS-PAGE after disulfide reduction
85000
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SDS-PAGE under nonreducing conditions
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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2 * 43000, SDS-PAGE after disulfide reduction
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
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-
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structure in presence of the donor substrate UDP-glucuronic acid
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crystal structure of the enzyme at 2.3 A in the presence of the UDP, Mn2+ and Galbeta(1-3)Galbeta(1-4)Xyl
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the X-ray crystal structure reveals that GlcAT1 is a globular protein with two subdomains
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vapor diffusion hanging drop method, crystal structure of the enzyme in the presence of UDP and Galbeta(1-3)Gal(6-O-sufate)beta(1-4)Xyl(2-O-phosphate)beta1-O-Ser
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D194A
about 85% of wild-type activity
D194A/D195A
inactive mutant protein
D194A/D196A
inactive mutant protein
D194E
about 85% of wild-type activity
D195A
about 25% of wild-type activity
D195A/D196A
inactive mutant protein
D195E
about 30% of wild-type activity
D196A
about 30% of wild-type activity
D196E
about 25% of wild-type activity
H308A
mutation abrogates the activity towards UDP-GlcA
H308R
mutation induces a major change in specificity. In contrast to wild-type enzyme the mutant is able to efficiently transfer Glc from UDP-Glc onto acceptor substrate Galbeta(1-3)Gal. The mutant enzyme remains able to catalyze the transfer of GlcA from UDP-GlyA onto Galbeta(1-3)Gal. UDP-GlcNAc is used at about the same rate as UDP-Glc. UDP-Gal is a weak donor substrate. UDP-Man is efficiently used as cosubstrate. No activity with GDP-Man
H308R/R277A
mutant enzyme shows no activity with UDP-GlcA as donor substrate, mutant enzyme is active with UDP-Gly as donor
C301A
-
mutant is not N-glycosylated, molecular weight is about 4000 Da less than that of the wild-type protein, enzyme is completely inactive
C33A
-
mutation abolishes the ability of the protein to form dimers
D113A
-
inactive mutant enzyme. Mutant enzyme is produced in a slightly lower amount compared with the wild-type enzyme
D113E
-
KM-value for UDP-glucuronate is 3.2fold higher than wild-type value
D113N
-
KM-value for UDP-glucuronate is 12.7fold higher than wild-type value
G222A
activity towards Galbeta(1-3)Galbeta1-O-methoxyphenyl is reduced 3.7fold and activity towards Galbeta(1-3)Gal(6-sulfate)beta1-O-methoxyphenyl is reduced 7.1fold
G222A/G223A
nearly complete loss of activity towards Galbeta(1-3)Galbeta1-O-methoxyphenyl and Galbeta(1-3)Gal(6-sulfate)beta1-O-methoxyphenyl
G223a
nearly complete loss of activity towards Galbeta(1-3)Galbeta1-O-methoxyphenyl and Galbeta(1-3)Gal(6-sulfate)beta1-O-methoxyphenyl
K317A
complete loss of activity towards Galbeta(1-3)Galbeta1-O-methoxyphenyl and Galbeta(1-3)Gal(6-sulfate)beta1-O-methoxyphenyl
K317R
activity towards Galbeta(1-3)Galbeta1-O-methoxyphenyl is reduced 7.2fold and activity towards Galbeta(1-3)Gal(6-sulfate)beta1-O-methoxyphenyl is reduced 6.5fold
Q318A
activity towards Galbeta(1-3)Galbeta1-O-methoxyphenyl is reduced 1.4fold and activity towards Galbeta(1-3)Gal(6-sulfate)beta1-O-methoxyphenyl is nearly identical to wild-type enzyme
R156A
-
inactive mutant enzyme
R156K
-
inactive mutant enzyme
R161A
-
inactive mutant enzyme
R161K
-
KM-value for UDP-glucuronate is 21.2fold higher than wild-type value
R310A
-
KM-value for UDP-glucuronate is 3.5fold higher than wild-type value
R310K
-
KM-value for UDP-glucuronate is identical to wild-type value
R310Q
-
KM-value for UDP-glucuronate is 1.5fold lower than wild-type value
W243A
inactive mutant protein
W243F
activity towards Galbeta(1-3)Galbeta1-O-methoxyphenyl is reduced 12fold and activity towards Galbeta(1-3)Gal(6-sulfate)beta1-O-methoxyphenyl is reduced 29fold
Y84A
-
inactive mutant enzyme, slightly less expressed than wild-type enzyme
Y84F
-
mutant enzyme shows 60% of wild-type activity. Mutant enzyme is produced at higher level than the wild-type protein. KM-value for UDP-glucuronate is 3fold higher than wild-type value
Y84H
-
mutant retains 21% of the activity with a KM value double that of the wild type. KM-value for UDP-glucuronate is 2.3fold higher than wild-type value
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Sepharose affinity chromatography
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Pichia pastoris
GlcAT-I cDNA probe are subcloned into Bluescript plasmid vectors and sequenced. Localization of GlcAT-1 and the pseudogene to two distinct chromosomes, 11q12-q13
expression in COS-1 cell, a soluble form of GlcAT-I generated by replacing the first 43 amino acids of GlcAT-I with a cleavable insulin signal sequence and a protein A IgG-binding domain
-
expression in Cos-1 cells
expression in Pichia pastoris
expression of full-length GlcAT-I in COS-1 cells
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Pedersen, L.C.; Darden, T.A.; Negishi, M.
Crystal structure of beta 1,3-glucuronyltransferase I in complex with active donor substrate UDP-GlcUA
J. Biol. Chem.
277
21869-21873
2002
Homo sapiens
Manually annotated by BRENDA team
Tone, Y.; Kitagawa, H.; Imiya, K.; Oka, S.; Kawasaki, T.; Sugahara, K.
Characterization of recombinant human glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans
FEBS Lett.
459
415-420
1999
Homo sapiens
Manually annotated by BRENDA team
Pedersen, L.C.; Tsuchida, K.; Kitagawa, H.; Sugahara, K.; Darden, T.A.; Negishi, M.
Heparan/chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I
J. Biol. Chem.
275
34580-34585
2000
Homo sapiens
Manually annotated by BRENDA team
Ouzzine, M.; Gulberti, S.; Netter, P.; Magdalou, J.; Fournel-Gigleux, S.
Structure/function of the human Ga1beta1,3-glucuronosyltransferase. Dimerization and functional activity are mediated by two crucial cysteine residues
J. Biol. Chem.
275
28254-28260
2000
Homo sapiens
Manually annotated by BRENDA team
Kitagawa, H.; Tone, Y.; Tamura, J.; Neumann, K.W.; Ogawa, T.; Oka, S.; Kawasaki, T.; Sugahara, K.
Molecular cloning and expression of glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans
J. Biol. Chem.
273
6615-6618
1998
Homo sapiens (O94766), Homo sapiens
Manually annotated by BRENDA team
Negishi, M.; Dong, J.; Darden, T.A.; Pedersen, L.G.; Pedersen, L.C.
Glucosaminylglycan biosynthesis: what we can learn from the X-ray crystal structures of glycosyltransferases GlcAT1 and EXTL2
Biochem. Biophys. Res. Commun.
303
393-398
2003
Homo sapiens
Manually annotated by BRENDA team
Kitagawa, H.; Taoka, M.; Tone, Y.; Sugahara, K.
Human glycosaminoglycan glucuronyltransferase I gene and a related processed pseudogene: genomic structure, chromosomal mapping and characterization
Biochem. J.
358
539-546
2001
Homo sapiens (Q9P2W7), Homo sapiens
Manually annotated by BRENDA team
Ouzzine, M.; Gulberti, S.; Levoin, N.; Netter, P.; Magdalou, J.; Fournel-Gigleux, S.
The donor substrate specificity of the human beta1,3-glucuronosyltransferase I toward UDP-glucuronic acid is determined by two crucial histidine and arginine residues
J. Biol. Chem.
277
25439-25445
2002
Homo sapiens (Q9P2W7), Homo sapiens
Manually annotated by BRENDA team
Gulberti, S.; Fournel-Gigleux, S.; Mulliert, G.; Aubry, A.; Netter, P.; Magdalou, J.; Ouzzine, M.
The functional glycosyltransferase signature sequence of the human beta 1,3-glucuronosyltransferase is a XDD motif
J. Biol. Chem.
278
32219-32226
2003
Homo sapiens (Q9P2W7), Homo sapiens
Manually annotated by BRENDA team
Gulberti, S.; Lattard, V.; Fondeur, M.; Jacquinet, J.C.; Mulliert, G.; Netter, P.; Magdalou, J.; Ouzzine, M.; Fournel-Gigleux, S.
Phosphorylation and sulfation of oligosaccharide substrates critically influence the activity of human beta1,4-galactosyltransferase 7 (GalT-I) and beta1,3-glucuronosyltransferase I (GlcAT-I) involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans
J. Biol. Chem.
280
1417-1425
2005
Homo sapiens (O94766), Homo sapiens
Manually annotated by BRENDA team
Lattard, V.; Fondeur-Gelinotte, M.; Gulberti, S.; Jacquinet, J.C.; Boudrant, J.; Netter, P.; Magdalou, J.; Ouzzine, M.; Fournel-Gigleux, S.
Purification and characterization of a soluble form of the recombinant human galactose-beta1,3-glucuronosyltransferase I expressed in the yeast Pichia pastoris
Protein Expr. Purif.
47
137-143
2006
Homo sapiens
Manually annotated by BRENDA team
Fondeur-Gelinotte, M.; Lattard, V.; Oriol, R.; Mollicone, R.; Jacquinet, J.C.; Mulliert, G.; Gulberti, S.; Netter, P.; Magdalou, J.; Ouzzine, M.; Fournel-Gigleux, S.
Phylogenetic and mutational analyses reveal key residues for UDP-glucuronic acid binding and activity of beta1,3-glucuronosyltransferase I (GlcAT-I)
Protein Sci.
15
1667-1678
2006
Homo sapiens
Manually annotated by BRENDA team
Tone, Y.; Pedersen, L.C.; Yamamoto, T.; Izumikawa, T.; Kitagawa, H.; Nishihara, J.; Tamura, J.; Negishi, M.; Sugahara, K.
2-o-phosphorylation of xylose and 6-o-sulfation of galactose in the protein linkage region of glycosaminoglycans influence the glucuronyltransferase-I activity involved in the linkage region synthesis
J. Biol. Chem.
283
16801-16807
2008
Homo sapiens
Manually annotated by BRENDA team