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Information on EC 4.2.2.19 - chondroitin B lyase and Organism(s) Pedobacter heparinus and UniProt Accession Q46079
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4.2.2.19 chondroitin B lyaseIUBMB Comments
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 . 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 .
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Word Map
- 4.2.2.19
- dermatan
- glycosaminoglycans
- proteoglycans
- disaccharide
- heparan
- heparinum
-
flavobacterium
- heparitinase
- hyaluronidase
- decorin
-
oversulfated
-
4-sulphate
-
nonsulfated
-
l-iduronic
- keratanase
-
4.2.2.5
-
idoa
- hexasaccharide
- galactosaminoglycans
- analysis
The taxonomic range for the selected organisms is: Pedobacter heparinus
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Reaction Schemes
Synonyms
chondroitinase b, chondroitin b lyase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
REACTION
REACTION DIAGRAM
COMMENTARY
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).
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).
acts on dermatan sulfate as sole substrate, active site structure involving Lys250, Arg271, His272, and Glu333, substrate binding mechanism involving Arg363 and Arg364
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).
acts on dermatan sulfate as sole substrate, catalytic mechanism, Glu333 is involved, substrate binding mechanism involving Arg318 and Arg364 interacting with the sulfate group
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).
acts on dermatan sulfate as sole substrate, enzyme cleaves the beta(1,4)-linkage of dermatan sulfate in a random manner, yielding 4,5-unsaturated dermatan sulfate disaccharides, calcium-dependent catalytic mechanism, active site structure
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
52227-83-5
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
dermatan sulfate
unsaturated dermatan sulfate disaccharide
-
structures
?
dermatan sulfate
unsaturated dermatan sulfate oligosaccharide
-
-
?
dermatan sulfate
unsaturated dermatan sulfate oligosaccharide
-
-
-
?
dermatan sulfate
?
analysis of interactions between enzyme and substrate
-
?
dermatan sulfate
?
dermatan sulfate purified from marine clam Scapharca inaequivalvis
-
-
?
additional information
?
-
-
digestion of amyloid fibrils with chondroitinase B causes a decrease in orthochromasy and anisotropy in comparison with untreated sections
-
-
?
additional information
?
-
-
rat bone marrow derived osteoblastic cells are cultured on either a machined titanium disk or an acid-etched titanium disk. At day 21 of culture chondroitinase B is added. For both the machined and acid-etched surface cultures, administration of the enzyme reduces the interfacial strength by 2530% compared with the untreated control cultures
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-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
dependent on, required for catalysis, activates, protein-Ca2+-oligosaccharide complex
additional information
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
glucose
represses enzyme induction in cells by chondroitin sulfate A
additional information
degree of inhibition by fatty acids depends on the chain length and the number of unsaturated bonds as well as the stereochemistry, overview
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0012
dermatan sulfate
recombinant wild-type enzyme, pH 8.0, 30°C, in presence of 5 mM Ca2+
0.0028 - 0.0046
dermatan sulfate
recombinant enzyme, pH 8.0, 30°C, dependent on expression system
0.0043
dermatan sulfate
recombinant wild-type enzyme, pH 8.0, 30°C, in presence of 0.01 mM Ca2+
additional information
additional information
kinetics of wild-type and mutant enzymes
-
additional information
additional information
-
kinetics of wild-type and mutant enzymes
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
87 - 190
dermatan sulfate
recombinant enzyme, pH 8.0, 30°C, dependent on expression system
210
dermatan sulfate
recombinant wild-type enzyme, pH 8.0, 30°C, in presence of 0.01 mM Ca2+
410
dermatan sulfate
recombinant wild-type enzyme, pH 8.0, 30°C, in presence of 5 mM Ca2+
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
-
analytical method for the determination of disaccharides derived from keratan, heparan, and dermatan sulfates in human serum and plasma by high-performance liquid chromatography/turbo ionspray ionization tandem mass spectrometry
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
i.e. Cytophaga heparina, Sphingobacterium heparinum, or Pedobacter heparinus, gene cslB
SwissProt
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
glycoprotein
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
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
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
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
PCR site-directed mutagenesis, E333 is a key residue in catalysis, reduced activity
H272A
PCR site-directed mutagenesis, H272 is a key residue in catalysis, reduced activity
N213Q
site-directed mutagenesis, Ca2+-binding residue, about 6fold reduced activity compared to the wild-type enzyme
R271A
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
PCR site-directed mutagenesis, R363 is a key residue in catalysis, 2fold increased activity
R364A
PCR site-directed mutagenesis, highly reduced activity, altered product profil, residue is involved in determination of substrate specificity
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
protease inhibitors like aprotinin, leupeptin or E64 are required for stability of the enzyme during storage at 277 K
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme comprising residues 25-506 overexpressed in Flavobacterium heparinum cells, to homogeneity
recombinant His-tagged enzyme from Escherichia coli, His-tag is removed, purified to homogeneity, 69fold
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli as N-terminally His-tagged enzyme, 2 different expression systems
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
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
analysis
method for quantitative detection of dermatan sulfate in heparin. Selective detection of deramtan sulfate in heparin is possible based on the increased value of UV absorption at 232 nm resulting from exhaustive depolymerization of dermatan sulfate. The first method facilitates analysis in heparin with dermatan sulfate concentrations greater than 0.1 mg/ml at 232 nm, with good linearity, precision and recovery. The second method allows sensitive and accurate detection of dermatan sulfate at concentrations below 0.1 mg/ml, with good linearity, precision and recovery. The linear range of dermatan sulfate detection is between 0.01 and 0.5 mg/ml
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Gu, K.; Linhardt, R.L.; Laliberte, M.; Gu, K.; Zimmermann, J.
Purification, characterization and specificity of chondroitin lyases and glycuronidase from Flavobacterium heparinum
Biochem. J.
312
569-577
1995
Pedobacter heparinus
Li, Y.; Matte, A.; Su, H.; Cygler, M.
Crystallization and preliminary X-ray analysis of chondroitinase B from Flavobacterium heparinum
Acta Crystallogr. Sect. D
55
1055-1057
1999
Pedobacter heparinus (Q46079), Pedobacter heparinus
Tkalec, A.L.; Fink, D.; Blain, F.; Zhang-Sun, G.; Laliberte, M.; Bennett, D.C.; Gu, K.; Zimmermann, J.J.F.; Su, H.
Isolation and expression in Escherichia coli of cslA and cslB, genes coding for the chondroitin sulfate-degrading enzymes chondroitinase AC and chondroitinase B, respectively, from Flavobacterium heparinum
Appl. Environ. Microbiol.
66
29-35
2000
Pedobacter heparinus (Q46079), Pedobacter heparinus
Pojasek, K.; Shriver, Z.; Kiley, P.; Venkataraman, G.; Sasisekharan, R.
Recombinant expression, purification, and kinetic characterization of chondroitinase AC and chondroitinase B from Flavobacterium heparinum
Biochem. Biophys. Res. Commun.
286
343-351
2001
Pedobacter heparinus (Q46079), Pedobacter heparinus
Ototani, N.; Yosizawa, Z.
Purification of chondroitinase B and chondroitinase C using glycosaminoglycan-bound AH-Sepharose 4B
Carbohydr. Res.
70
295-306
1979
Pedobacter heparinus
Pojasek, K.; Raman, R.; Kiley, P.; Venkataraman, G.; Sasisekharan, R.
Biochemical characterization of the chondroitinase B active site
J. Biol. Chem.
277
31179-31186
2002
Pedobacter heparinus (Q46079), Pedobacter heparinus
Michel, G.; Pojasek, K.; Li, Y.; Sulea, T.; Linhardt, R.J.; Raman, R.; Prabhakar, V.; Sasisekharan, R.; Cygler, M.
The structure of chondroitin B lyase complexed with glycosaminoglycan oligosaccharides unravels a calcium-dependent catalytic machinery
J. Biol. Chem.
279
32882-32896
2004
Pedobacter heparinus (Q46079), Pedobacter heparinus
Suzuki, K.; Terasaki, Y.; Uyeda, M.
Inhibition of hyaluronidases and chondroitinases by fatty acids
J. Enzyme Inhib. Med. Chem.
17
183-186
2002
Pedobacter heparinus (Q46079)
Huang, W.; Matte, A.; Li, Y.; Kim, Y.S.; Linhardt, R.J.; Su, H.; Cygler, M.
Crystal structure of chondroitinase B from Flavobacterium heparinum and its complex with a disaccharide product at 1.7 A resolution
J. Mol. Biol.
294
1257-1269
1999
Pedobacter heparinus (Q46079), Pedobacter heparinus
Makovitzky, J.; Richter, S.; Appel, T.R.
Topooptical investigations and enzymatic digestions on tissue-isolated amyloid fibrils
Acta Histochem.
108
193-196
2006
Pedobacter heparinus
Oguma, T.; Tomatsu, S.; Montano, A.M.; Okazaki, O.
Analytical method for the determination of disaccharides derived from keratan, heparan, and dermatan sulfates in human serum and plasma by high-performance liquid chromatography/turbo ionspray ionization tandem mass spectrometry
Anal. Biochem.
368
79-86
2007
Pedobacter heparinus
Nakamura, H.; Shim, J.; Butz, F.; Aita, H.; Gupta, V.; Ogawa, T.
Glycosaminoglycan degradation reduces mineralized tissue-titanium interfacial strength
J. Biomed. Mater. Res. A
77
478-486
2006
Pedobacter heparinus
Volpi, N.; Maccari, F.
Structural characterization and antithrombin activity of dermatan sulfate purified from marine clam Scapharca inaequivalvis
Glycobiology
19
356-367
2008
Pedobacter heparinus (Q46079), Pedobacter heparinus
Wu, J.; Ji, Y.; Su, N.; Li, Y.; Liu, X.; Mei, X.; Zhou, Q.; Zhang, C.; Xing, X.H.
Establishment of chondroitin B lyase-based analytical methods for sensitive and quantitative detection of dermatan sulfate in heparin
Carbohydr. Polym.
144
338-345
2016
Pedobacter heparinus (Q46079), Pedobacter heparinus DSM 2366 (Q46079)
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