Information on EC 2.4.1.135 - galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.4.1.135
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RECOMMENDED NAME
GeneOntology No.
galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
UDP-alpha-D-glucuronate + [protein]-3-O-(beta-D-galactosyl-(1->3)-beta-D-galactosyl-(1->4)-beta-D-xylosyl)-L-serine = UDP + [protein]-3-O-(beta-D-GlcA-(1->3)-beta-D-Gal-(1->3)-beta-D-Gal-(1->4)-beta-D-Xyl)-L-serine
show the reaction diagram
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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
Glycosaminoglycan biosynthesis - chondroitin sulfate / dermatan sulfate
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Glycosaminoglycan biosynthesis - heparan sulfate / heparin
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glycosaminoglycan-protein linkage region biosynthesis
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Mannose type O-glycan biosynthesis
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Metabolic pathways
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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
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9030-08-4
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene B3GAT3
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
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the enzyme is a key regulator and rate-limiting enzyme for the synthesis of GAG chains
physiological function
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the enzyme mediates a glucuronyl transfer reaction during the biosynthesis of the carbohydrate epitope HNK-1, human natural killer-1, also known as CD57, a marker of NK cells
additional information
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gene B3GAT3 is a unigene, pathway enrichment analysis of assembled unigenes, overview. Identification of markers of NK cells. Enzyme structure modelling using the structure of human GlcAT-P as a template
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
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
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-
-
-
?
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
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-
-
-
?
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
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-
-
-
?
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
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?
UDP-Gal + Galbeta(1-3)Gal
Galbeta(1-3)Galbeta(1-3)Gal + UDP
show the reaction diagram
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no activity with wild-type enzyme, weak activity with mutant enzyme H308R
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-
?
UDP-galacturonic acid + Galbeta(1-3)Gal
Galbeta(1-3)Galbeta(1-3)Gal + UDP
show the reaction diagram
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-
-
?
UDP-GlcNAc + Galbeta(1-3)Gal
GlcNAcbeta(1-3)Galbeta(1-3)Gal + UDP
show the reaction diagram
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no activity with wild-type enzyme, activity with mutant enzyme H308R is nearly equal to activity with UDP-Glc
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-
?
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
UDP-glucuronate + Galbeta(1-3)Gal
GlcAbeta(1-3)Galbeta(1-3)Gal + UDP
show the reaction diagram
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?
UDP-glucuronate + Galbeta(1-3)Gal(6-sulfate)beta1-O-methoxyphenyl
UDP + ?
show the reaction diagram
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-
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?
UDP-glucuronate + Galbeta(1-3)Gal-O-benzyl
UDP + ?
show the reaction diagram
best substrate
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?
UDP-glucuronate + Galbeta(1-3)Gal-O-naphthalenemethanol
UDP + ?
show the reaction diagram
best substrate
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-
?
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)Galbeta(1-4)Xyl-1-O-Ser
UDP + GlcAbeta(1-3)Galbeta(1-3)Galbeta(1-4)Xyl-1-O-Ser
show the reaction diagram
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?
UDP-glucuronate + Galbeta(1-3)Galbeta1-O-methoxyphenyl
UDP + ?
show the reaction diagram
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?
UDP-glucuronate + Galbeta(1-3)GlcNAcalpha-O-benzyl
UDP + ?
show the reaction diagram
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?
UDP-glucuronate + Galbeta(1-3)GlcNAcalpha-O-naphthalenemethanol
UDP + ?
show the reaction diagram
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?
UDP-glucuronate + Galbeta(1-3)GlcNAcbeta-O-naphthalenemethanol
UDP + ?
show the reaction diagram
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?
UDP-glucuronate + Galbeta(1-4)Glcbeta-O-naphthalenemethanol
UDP + ?
show the reaction diagram
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?
UDP-glucuronate + Galbeta(1-4)GlcNAcbeta-O-naphthalenemethanol
UDP + ?
show the reaction diagram
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?
UDP-glucuronate + Galbeta-O-1-naphthol
UDP + GlcAbeta(1-3)Galbeta-O-1-naphthol
show the reaction diagram
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?
UDP-glucuronate + Galbeta-O-2-naphthol
UDP + GlcAbeta(1-3)Galbeta-O-2-naphthol
show the reaction diagram
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?
UDP-glucuronate + Galbeta1-3Gal
UDP + Galbeta1-3Galbeta1-3Gal
show the reaction diagram
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the enzyme exhibits a strict selectivity towards Galbeta1-3Gal structures
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?
UDP-Man + Galbeta(1-3)Gal
Manbeta(1-3)Galbeta(1-3)Gal + UDP
show the reaction diagram
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no activity with wild-type enzyme, efficient reaction with mutant enzyme H308R
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?
UDPglucuronate + 3-beta-D-galactosyl-4-beta-D-galactosyl-O-beta-D-xylosylprotein
?
show the reaction diagram
UDPglucuronate + 3-beta-D-galactosyl-D-galactose
UDP + 3-beta-D-glucuronosyl-3-beta-D-galactosyl-D-galactose
show the reaction diagram
UDPglucuronate + 3-beta-D-galactosyl-D-galactosyl-4-xylose
UDP + 3-beta-D-glucuronosyl-3-beta-D-galactosyl-D-galactosyl-4-xylose
show the reaction diagram
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?
UDPglucuronate + 3-beta-D-galactosyl-D-galactosyl-4-xylosylserine
UDP + 3-beta-D-glucuronosyl-3-beta-D-galactosyl-D-galactosyl-4-xylosylserine
show the reaction diagram
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?
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-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
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plays a key role in glycosaminoglycan biosynthesis
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?
UDP-glucuronate + Galbeta(1-3)Galbeta(1-4)Xyl
UDP + GlcAbeta(1-3)-Galbeta(1-3)Galbeta(1-4)Xyl
show the reaction diagram
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high expression of the GlcAT-I gene renders the cells capable of synthesizing the HNK-1 epitope
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?
UDP-glucuronate + Galbeta(1-3)Galbeta(1-4)Xyl-1-O-Ser
UDP + GlcAbeta(1-3)Galbeta(1-3)Galbeta(1-4)Xyl-1-O-Ser
show the reaction diagram
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?
UDPglucuronate + 3-beta-D-galactosyl-4-beta-D-galactosyl-O-beta-D-xylosylprotein
?
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
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stimulation at 10 mM is 25% of the stimulation with Mn2+. No further activation when the concentration is increased up to 50 mM
Cu2+
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divalent cations essential, 2.7% as effective as Mn2+
Zn2+
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stimulation at 10 mM is 50% of the stimulation with Mn2+. No further activation when the concentration is increased up to 50 mM
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,3-Butanedione
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irreversible. UDP-glucuronic acid protects, no protection by UDP-glucose. Activity of H308R towards UDP-glucose is unaffected by the reagent
3-O-beta-D-Galactosyl-galactose
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with 3-galactosyl-4-galactosyl-xylose as substrate
N-Phenylmaleimide
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additional information
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no inhibition by chondroitin 6-sulfate trisaccharides or pentasaccharides containing N-acetylglucosamine 6-sulfate at their non-reducing termini
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0091
3-beta-galactosyl-galactose
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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, 37C
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, 37C
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, 37C
0.046
Galbeta(1-3)Galbeta(1-4)Xylbeta1-O-Ser
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pH 6.5, 2 mM MnCl2, 37C
0.67
Galbeta1,3Galbeta-O-naphthalenemethanol
pH 6.5, 37C
3.2
Galbeta1,3GalNAcbeta-O-naphthalenemethanol
pH 6.5, 37C
2.9
Galbeta1,3GlcNAcbeta-O-naphthalenemethanol
pH 6.5, 37C
1.8
Galbeta1,4GlcNAcbeta-O-naphthalenemethanol
pH 6.5, 37C
4.48
Galbeta1-3Gal
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37C, pH 6.5
0.233
UDP-Glc
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pH 5.0, 37C, mutant enzyme H308R
0.0499 - 0.059
UDP-GlcA
0.108
UDP-GlcNAc
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pH 5.0, 37C, mutant enzyme H308R
0.04 - 1.27
UDP-glucuronate
0.074
UDP-Man
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pH 5.0, 37C, mutant enzyme H308R
0.00025 - 0.287
UDPglucuronate
additional information
additional information
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 7.5
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 8.5
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about 50% of maximal activity at pH 6.5 and pH 8.5
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
85000
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SDS-PAGE under nonreducing conditions
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 43000, SDS-PAGE
dimer
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2 * 43000, SDS-PAGE after disulfide reduction
additional information
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determination of enzyme tertiary structure by homology modelling using the human enzyme structure, PDB ID 1v83
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
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
partial
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Sepharose affinity chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
chimeric proteins composed of mutant GlcATI fused to IgG binding domain of protein A or to green fluoresecent protein do not yield the protein at the expected mass
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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
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expression in Cos-1 cells
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expression in Pichia pastoris
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expression of full-length GlcAT-I in COS-1 cells
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gene B3GAT3, a unigene, DNA and amino acid sequence determination and analysis, sequence comparison with the human enzyme and phylogenetic analysis
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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
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transfection of a CHO cell mutant defective in GlcUAT-I with the hamster cDAN restores glycosaminoglycan assembly in vivo
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
GlcAT-I promoter activity is suppressed by co-expression of calcineurin and nuclear factors of activated T cells 2, 3, and 4, treatment with WP631 at 50-100 nM completely abolishes ionomycin-mediated induction in GlcAT-I-D reporter activity as well as suppressing basal promoter activity
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the enzyme is upregulated 3fold by 5 ng/ml transforming growth factor-beta1. Enzyme expression is increased in idiopathic pulmonary fibrosis, fibrotic rat lungs, and fibrotic lung fibroblasts
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treatment with 0.001 mM ionomycin for 24 h results in increased GlcAT-I expression, ionomycin mediated induction of GlcAT-I promoter activity requires TonEBP binding to TonE, calcium regulates GlcAT-I expression in cells of the nucleus pulposus
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treatment with both BMP-2 (200 ng/ml) and TGFbeta (10 ng/ml) for 24 h results in increased GlcAT-I mRNA levels in nucleus pulposus cells, however, simultaneous treatment of nucleus pulposus cells with TGFbeta and BMP-2 does not synergize GlcAT-I mRNA expression. GlcAT-I regulation is mediated through transcription factors AP1, Sp1, and TonEBP (NFAT5). Treatment with MAPK inhibitors blocks BMP-2 and TGFbeta induced GlcAT-I expression. p38delta is important in GlcAT-I activation
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under hypoxic conditions (1% O2) there is an increase in enzyme expression both in nucleus pulposus cells and in N1511 chondrocytes. Suppression of hypoxia-inducible factor-1alpha and hypoxia-inducible factor-2alpha induces enzyme promoter activity and expression only in nucleus pulposus cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
V111M
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mutation renders glycosaminoglycan biosynthesis temperature-sensitive, greater synthesis at 33C compared with 37C
C301A
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mutant is not N-glycosylated, molecular weight is about 4000 Da less than that of the wild-type protein, enzyme is completely inactive
C33A
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mutation abolishes the ability of the protein to form dimers
D113A
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inactive mutant enzyme. Mutant enzyme is produced in a slightly lower amount compared with the wild-type enzyme
D113E
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KM-value for UDP-glucuronate is 3.2fold higher than wild-type value
D113N
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KM-value for UDP-glucuronate is 12.7fold higher than wild-type value
D194A
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about 85% of wild-type activity
D194A/D195A
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inactive mutant protein
D194A/D196A
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inactive mutant protein
D194E
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about 85% of wild-type activity
D195A
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about 25% of wild-type activity
D195A/D196A
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inactive mutant protein
D195E
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about 30% of wild-type activity
D196A
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about 30% of wild-type activity
D196E
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about 25% of wild-type activity
G222A
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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
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nearly complete loss of activity towards Galbeta(1-3)Galbeta1-O-methoxyphenyl and Galbeta(1-3)Gal(6-sulfate)beta1-O-methoxyphenyl
G223a
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nearly complete loss of activity towards Galbeta(1-3)Galbeta1-O-methoxyphenyl and Galbeta(1-3)Gal(6-sulfate)beta1-O-methoxyphenyl
H308A
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mutation abrogates the activity towards UDP-GlcA
H308R
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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
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mutant enzyme shows no activity with UDP-GlcA as donor substrate, mutant enzyme is active with UDP-Gly as donor
K317A
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complete loss of activity towards Galbeta(1-3)Galbeta1-O-methoxyphenyl and Galbeta(1-3)Gal(6-sulfate)beta1-O-methoxyphenyl
K317R
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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
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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
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inactive mutant enzyme
R156K
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inactive mutant enzyme
R161A
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inactive mutant enzyme
R161K
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KM-value for UDP-glucuronate is 21.2fold higher than wild-type value
R310A
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KM-value for UDP-glucuronate is 3.5fold higher than wild-type value
R310K
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KM-value for UDP-glucuronate is identical to wild-type value
R310Q
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KM-value for UDP-glucuronate is 1.5fold lower than wild-type value
W243A
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inactive mutant protein
W243F
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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
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inactive mutant enzyme, slightly less expressed than wild-type enzyme
Y84F
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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
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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
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