Information on EC 4.2.2.20 - chondroitin-sulfate-ABC endolyase

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

EC NUMBER
COMMENTARY hide
4.2.2.20
-
RECOMMENDED NAME
GeneOntology No.
chondroitin-sulfate-ABC endolyase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Endolytic cleavage of (1->4)-beta-galactosaminic bonds between N-acetylgalactosamine and either D-glucuronic acid or L-iduronic acid to produce a mixture of Delta4-unsaturated oligosaccharides of different sizes that are ultimately degraded to Delta4-unsaturated tetra- and disaccharides
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
chondroitin sulfate degradation I (bacterial)
-
-
dermatan sulfate degradation I (bacterial)
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-
SYSTEMATIC NAME
IUBMB Comments
chondroitin-sulfate-ABC endolyase
This enzyme degrades a variety of glycosaminoglycans of the chondroitin-sulfate- and dermatan-sulfate type. Chondroitin sulfate, chondroitin-sulfate proteoglycan and dermatan sulfate are the best substrates but the enzyme can also act on hyaluronan at a much lower rate. Keratan sulfate, heparan sulfate and heparin are not substrates. In general, chondroitin sulfate (CS) and dermatan sulfate (DS) chains comprise a linkage region, a chain cap and a repeat region. The repeat region of CS is a repeating disaccharide of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) [-4)GlcA(beta1-3)GalNAc(beta1-]n, which may be O-sulfated on the C-4 and/or C-6 of GalNAc and C-2 of GlcA. GlcA residues of CS may be epimerized to iduronic acid (IdoA) forming the repeating disaccharide [-4)IdoA(alpha1-3)GalNAc(beta1-]n of DS. Both the concentrations and locations of sulfate-ester substituents vary with glucosaminoglycan source [5]. The related enzyme EC 4.2.2.21, chondroitin-sulfate-ABC exolyase, has the same substrate specificity but removes disaccharide residues from the non-reducing ends of both polymeric chondroitin sulfates and their oligosaccharide fragments produced by EC 4.2.2.20 [4].
CAS REGISTRY NUMBER
COMMENTARY hide
9024-13-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
chondroitin
?
show the reaction diagram
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32% of the rate with chondroitin-6-sulfate
-
-
?
chondroitin 4-sulfate
?
show the reaction diagram
chondroitin 6-sulfate
?
show the reaction diagram
-
-
-
-
?
chondroitin sulfate
?
show the reaction diagram
-
-
-
-
?
chondroitin sulfate A
?
show the reaction diagram
-
-
-
-
?
chondroitin sulfate C
?
show the reaction diagram
-
-
-
-
?
chondroitin sulfate D
?
show the reaction diagram
-
70% of the rate with chondroitin-6-sulfate
-
-
?
chondroitin sulfate E
?
show the reaction diagram
-
23% of the rate with chondroitin-6-sulfate
-
-
?
chondroitin sulfate proteoglycan
?
show the reaction diagram
-
70% of the rate with chondroitin-6-sulfate
-
-
?
chondroitin-6-sulfate
unsaturated sulfated disaccharides
show the reaction diagram
-
-
-
-
?
dermatan sulfate
?
show the reaction diagram
dermatan sulfate
unsaturated sulfated disaccharide + oligosaccharides
show the reaction diagram
-
-
-
-
?
hyaluronan
?
show the reaction diagram
-
1% of the rate with chondroitin-6-sulfate
-
-
?
additional information
?
-
-
no substrate: keratan sulfate, heparin, heparan sulfate. Enzyme produces a wide range of differently sized oligosaccharides. After exhaustive degradation, end products are tetra- and disaccharides
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-
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cu2+
-
1 mM, 80% inhibition
Fe2+
-
1 mM, 80% inhibition
Ni2+
-
1 mM, 80% inhibition
Zn2+
-
1 mM, complete inhibition
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.031 - 0.05
chondroitin 4-sulfate
0.055
chondroitin 6-sulfate
-
pH 6.8, 37°C
0.073
chondroitin sulfate A
-
recombinant protein fused to maltose-binding protein, pH 7.4, 50°C
0.066
chondroitin-6-sulfate
-
pH 8.0, 37°C
0.094
dermatan sulfate
-
pH 6.8, 37°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
69.7 - 89
chondroitin 4-sulfate
100
chondroitin 6-sulfate
Proteus vulgaris
-
pH 6.8, 37°C
587
chondroitin sulfate A
Proteus vulgaris
-
recombinant protein fused to maltose-binding protein, pH 7.4, 50°C
48.5
dermatan sulfate
Proteus vulgaris
-
pH 6.8, 37°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1600 - 2800
chondroitin 4-sulfate
2137
1800
chondroitin 6-sulfate
Proteus vulgaris
-
pH 6.8, 37°C
1675
1517
dermatan sulfate
Proteus vulgaris
-
pH 6.8, 37°C
907
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
310
-
pH 8.0, 37°C
3180
-
recombinant protein fused to maltose-binding protein, pH 7.4, 50°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2
-
assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.7
-
degeneration of protein above
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
-
and 8.25, isoelectric focusing
8.25
-
and 8.0, isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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immature articular cartilage explants from the superficial and middle layers. Manipulation of glycosaminoglycan content can distinctly alter the growth phenotype of cartilage
Manually annotated by BRENDA team
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sciatic nerve transsection and end-to-end repair, with one nerve injected with chondroitinase ABC and the contralateral nerve treated with vehicle alone
Manually annotated by BRENDA team
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
210 min, 78% residual activity
40
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30 min, 47% loss of activty
47
-
melting temperature
49
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melting temperature, presence of 20% glycerol
52
-
melting temperature, presence of 1 M sorbitol
54
-
melting temperature, presence of 1 M trehalose
additional information
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in commercial preparations, there is a significant effect of lot and time on thermal stability. Average enzymatic activity is significantly decreased after 1 and 3 days at 39°C and 37°C, resp. The average activity seen after 1 day is significantly different between the two temperatures. Addition of bovine serum albumin preserves enzymatic activity at 1 day, but not 3 days, at 39°C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the enzyme catalytic activity and intrinsic fluorescence intensity increase in the presence of cosolvents glycerol, sorbitol and trehalose whereas no considerable conformational changes are observed in far-UV CD spectra. In the presence of trehalose a significant increase in meltin temperature is observed.
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-18°C, 3 days, complete loss of activity
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25°C, 17 days, complete loss of activity, in presence of 1 M trehalose 85% residual activity after 21 days
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37°C, 10 days, presence of 1 M trehalose, 20% residual activity
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4°C, 21 days, 80% residual activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
isoform chondroitinase B, cell disruption by ultrasound, under mild conditions, solubilization of most of enzyme activity
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recombinant protein
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in HEK-293 cell
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fusion protein with maltose-binding protein, expression in Escherichia coli
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
when expressed in a mammalian cell, residues N675, N515, N345, N338 and N282 are all glycosylated which prevents secretion of functional enzyme. Directed mutagenesis of selected N-glycosylation sites allows efficient secretion of active chondroitinase. Mutation if residue N751 renders the enzyme inactive. Mutations required for efficient secretion of active enzyme are those eliminating glycosylation at N282, N336, N345, and N515, and mutation of N675 increases the activity still further
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Q140A
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mutation improves both activity and thermal stability of the enzyme, and additionally improves resistance to trypsin degradation
Q140G
-
mutation improves both activity and thermal stability of the enzyme, and additionally improves resistance to trypsin degradation
Q140N
-
mutation reduces the enzyme activity and destabilizes
additional information
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engineering of a mammalian expression system of an epitope-tagged chondroitinase ABC protein. At the 5'-end, an optimized KOZAK-ATG sequence is inserted, to allow efficient eukaryotic translation, followed by a 60 base pair secretion sequence from the fish Oreochromis aureus, engineered for efficient crosshost recombinant protein expression. At the 3'-end, a 3xFlag epitope tag is added. The addition of the eukaryotic secretion signal sequence allows secretion, but interferes with function of the secreted enzyme. In contrast, expression of the chondroitinase ABC gene without N-terminal eukaryotic secretion sequence or bacterial hydrophobic leader sequence leads to efficient secretion of a biologically active chondroitinase ABC protein from both immortalized and primary cells. The C-terminal epitope tag can be utilized to follow expression of this protein
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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bond strength of two etch-and-rinse adhesives to chondroitinase ABC treated dentin is investigated. Human extracted molars are treated with chondroitinase ABC. Increased mean values of microtensile bond strength and reduced nanoleakage expression are shown for both adhesives after chondroitinase ABC treatment of the dentin surface. This study supports the hypothesis that adhesion can be enhanced by removal of chondroitin 4/6 sulfate and dermatan sulfate, probably due to a reduced amount of water content and enlarged interfibrillar spaces
medicine
molecular biology
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glycosaminoglycans are extracted from cooked haddock muscle. Reverse phase chromatography and digestion with chondroitinase ABC (Chase) is used. FeCl3 is mixed with the purified glycosaminoglycans, and Fe uptake is measured by ferritin formation using an in vitro digestion/Caco-2 cell model. The identificative analyses suggest that chondroitin/dermatan sulfate-related structures promote Fe uptake by Caco-2 cells