Information on EC 4.2.2.19 - chondroitin B lyase

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

EC NUMBER
COMMENTARY hide
4.2.2.19
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RECOMMENDED NAME
GeneOntology No.
chondroitin B lyase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Eliminative cleavage of dermatan sulfate containing (1->4)-beta-D-hexosaminyl and (1->3)-beta-D-glucurosonyl or (1->3)-alpha-L-iduronosyl linkages to disaccharides containing 4-deoxy-beta-D-gluc-4-enuronosyl groups to yield a 4,5-unsaturated dermatan-sulfate disaccharide (DeltaUA-GalNAc-4S).
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
elimination
-
C-O bond cleavage
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
dermatan sulfate degradation I (bacterial)
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-
SYSTEMATIC NAME
IUBMB Comments
chondroitin B lyase
This is the only lyase that is known to be specific for dermatan sulfate as substrate. The minimum substrate length required for catalysis is a tetrasaccharide [2]. 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].
CAS REGISTRY NUMBER
COMMENTARY hide
52227-83-5
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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
dermatan sulfate
?
show the reaction diagram
dermatan sulfate
disaccharides
show the reaction diagram
-
-
structures
?
dermatan sulfate
oligosaccharides
show the reaction diagram
-
-
-
?
dermatan sulfate
unsaturated oligosaccharides
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
dermatan sulfate
?
show the reaction diagram
-
substrate is involved in activities related to metastasis
-
?
additional information
?
-
-
enzyme inhibits endothelial and melanoma proliferation and invasion, but to a lesser extent than chondroitin AC lyase, EC 4.2.2.5, enzyme has no effect on gelatinase excretion by melanoma cells, enzyme does not activate caspase-3 activity and apoptosis in melanoma and endothelial cells
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
dependent on, required for catalysis, activates, protein-Ca2+-oligosaccharide complex
additional information
-
no bound Ca2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(13Z)-docos-13-enoic acid
-
strong inhibition
arachidic acid
-
-
behenic acid
-
-
capric acid
-
-
EGTA
complete inhibition
eicosadienoic acid
-
-
eicosanoic acid
-
strong inhibition
eicosapentaenoic acid
-
-
eicosatetraenoic acid
-
-
eicosatrienoic acid
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; nonconmpetitive
elaidic acid
-
strong inhibition
glucose
represses enzyme induction in cells by chondroitin sulfate A
lauric acid
-
-
linoleic acid
-
strong inhibition
linolenic acid
-
strong inhibition
methyl (9Z)-octadecenoate
-
-
myristic acid
-
-
myristoleic acid
-
-
nervonic acid
-
strong inhibition
oleic acid
-
strong inhibition
palmitic acid
-
-
palmitoleic acid
-
-
petroselinic acid
-
strong inhibition
ricinoleic acid
-
-
stearic acid
-
-
vaccenic acid
-
strong inhibition
additional information
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degree of inhibition by fatty acids depends on the chain length and the number of unsaturated bonds as well as the stereochemistry, overview
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
chondroitin sulfate A
induces enzyme expression in cells
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0012 - 0.0163
dermatan sulfate
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4.6 - 410
dermatan sulfate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.037
eicosatrienoic acid
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pH 8.0, 37°C
additional information
additional information
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-
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
80.8
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purified recombinant enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8 - 8
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broad optimum
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9
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isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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melanoma cell line
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Pedobacter heparinus (strain ATCC 13125 / DSM 2366 / CIP 104194 / JCM 7457 / NBRC 12017 / NCIMB 9290 / NRRL B-14731 / HIM 762-3)
Pedobacter heparinus (strain ATCC 13125 / DSM 2366 / CIP 104194 / JCM 7457 / NBRC 12017 / NCIMB 9290 / NRRL B-14731 / HIM 762-3)
Pedobacter heparinus (strain ATCC 13125 / DSM 2366 / CIP 104194 / JCM 7457 / NBRC 12017 / NCIMB 9290 / NRRL B-14731 / HIM 762-3)
Pedobacter heparinus (strain ATCC 13125 / DSM 2366 / CIP 104194 / JCM 7457 / NBRC 12017 / NCIMB 9290 / NRRL B-14731 / HIM 762-3)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
54070
-
mass spectrometry
55000
x * 55000, SDS-PAGE, recombinant and native enzyme
55200
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x * 55200, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
structural analysis
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
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a heptasaccharide glycosylation site attached to Ser234
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
6-8 mg/ml purified recombinant enzyme, residues 25-506, in 20 mM Tris-HCl, pH 8.0, 1 mM sodium phosphate, pH 7.0, 3 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 0.001 mg/ml aprotinin, 0.001 mg/ml leupeptin, 0.001 mg/ml E64, hanging drop vapour diffusion method, 292 K, mixed with a double volume of reservoir solution containing 19% w/v PEG 8000, 100 mM bicine buffer, pH 9.0, or 100 mM Tris-HCl, pH 8.8, 0.15 M ammonium acetate, 15% v/v 2-methyl-2,4-pentanediol, drops are suspended over 1 ml of resevroir solution, crystals appear overnight, seeds are introduced into drops conisting of 0.002 ml protein solution, 6.1 mg/ml protein, and 0.004 ml reservoir solution containing 16.5% w/v PEG 8000, 0.1 M Tris-HCl, pH 8.8, 15% v/v 2-methyl-2,4-pentanediol, 0.25 M ammonium acetate, 292 K, 2-3 weeks, X-ray diffraction structure determination and analysis at 2.20-2.28 A resolution
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purified recombinant enzyme, residues 25-506 corresponding to the full size mature enzyme, labeling and complexing of enzyme in crystals via soaking in cryoprotectant solution containing the heavy atom or disaccharide product in 22.5% w/v PEG 8000, 0.1 M Tris-HCl, pH 8.7, 15% v/v 2-methyl-2,4-pentanediol, and 0.25 M ammonium acetate, equilibration over 1 ml reservoir solution, complexed with disaccharide product, X-ray diffraction structure determination and analysis at 1.7 A resolution, structure modeling of the right-handed parallel beta-helix protein
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purified recombinant wild-type enzyme complexed with dermatan sulfate pentasaccharide and hexasaccharide, or chondroitin-4-sulfate tetrasacharide, X-ray diffraction structure determination and analysis at 1.7-1.8 A resolution, modeling of substrate binding in the active site groove
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
recombinant and native enzyme are completely stable
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
protease inhibitors like aprotinin, leupeptin or E64 are required for stability of the enzyme during storage at 277 K
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purification based on affinity chromatography
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recombinant enzyme comprising residues 25-506 overexpressed in Flavobacterium heparinum cells, to homogeneity
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recombinant His-tagged enzyme from Escherichia coli, His-tag is removed, purified to homogeneity, 69fold
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to homogeneity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli as N-terminally His-tagged enzyme, 2 different expression systems
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gene cslB, expression in Escherichia coli without the N-terminal signal sequence, different expression plasmids constructed, expression in soluble and insoluble form
overexpression of enzyme, residues 25-506, in Flavobacterium heparinum cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E243A
site-directed mutagenesis, Ca2+-binding residue, about 3.6fold reduced activity compared to the wild-type enzyme
E243A/E245A
site-directed mutagenesis, Ca2+-binding residue, about 4fold reduced activity compared to the wild-type enzyme
E245A
site-directed mutagenesis, Ca2+-binding residue, about 6fold reduced activity compared to the wild-type enzyme
E333A
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PCR site-directed mutagenesis, E333 is a key residue in catalysis, reduced activity
H272A
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PCR site-directed mutagenesis, H272 is a key residue in catalysis, reduced activity
K250A
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PCR site-directed mutagenesis, inactive mutant
N213Q
site-directed mutagenesis, Ca2+-binding residue, about 6fold reduced activity compared to the wild-type enzyme
R271A
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PCR site-directed mutagenesis, R271 is a key residue in catalysis
R271E
site-directed mutagenesis, active site mutant, catalytically inactive
R271K
site-directed mutagenesis, active site mutant, about 10fold reduced activity compared to the wild-type enzyme
R363A
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PCR site-directed mutagenesis, R363 is a key residue in catalysis, 2fold increased activity
R364A
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PCR site-directed mutagenesis, highly reduced activity, altered product profil, residue is involved in determination of substrate specificity