Information on EC 4.2.2.8 - heparin-sulfate lyase

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

EC NUMBER
COMMENTARY hide
4.2.2.8
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RECOMMENDED NAME
GeneOntology No.
heparin-sulfate lyase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
elimination of sulfate; appears to act on linkages between N-acetyl-D-glucosamine and uronate. Product is an unsaturated sugar.
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
elimination
-
-
-
-
elimination of sulfate
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
heparan sulfate degradation
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-
SYSTEMATIC NAME
IUBMB Comments
heparin-sulfate lyase
Does not act on N,O-desulfated glucosamine or N-acetyl-O-sulfated glucosamine linkages.
CAS REGISTRY NUMBER
COMMENTARY hide
37290-86-1
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain HJ-15
UniProt
Manually annotated by BRENDA team
strain HJ-15
UniProt
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
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differential effects of heparitinase I and heparitinase III, EC 4.2.2.7, on endothelial tube formation. The enzymes inhibit tube formation and reduce tumor-derived neovascularization in vivo by reducing bFGF binding and subsequent signaling, HepIII has a stronger effect than Hep I. Heparitinases, isolated from Flavobacterium heparinum, cleave heparan sulfate chains at defined locations, Hep I cleaves heparin sulfate chains containing glucuronic acid residues adjacent to glucosamine residues containing either N-acetyl or N-sulfate groups. Hep I generates fragments that retain their growth factor binding capability and therefore still potentiate tube formation
additional information
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intermolecular disulfide bond formation in HepI important for catalysis, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
beta-D-glucopyranosyluronic acid
?
show the reaction diagram
-
major requirement for enzyme action is reduced sulfation, cannot cleave linkages containing unsulfated GalAP residues
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-
?
chemically modified heparins
?
show the reaction diagram
-
-
-
-
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heparan sulfate
?
show the reaction diagram
heparan sulfate
n alpha-deltaUA-glcNAc
show the reaction diagram
heparan sulfate
unsaturated, non-sulfated di- and tetrasaccharides + SO42-
show the reaction diagram
heparin
?
show the reaction diagram
heparitin sulfates
?
show the reaction diagram
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and related compounds
-
-
?
heparosan polysaccharide
heparosan oligosaccharide
show the reaction diagram
-
-
-
-
?
hyaluronic acid
?
show the reaction diagram
N,6-sulfated glucosaminido-alpha-1,4-glucuronic acid oligosaccharide
N-sulfated glucosaminido-alpha-1,4-glucuronic acid oligosaccharide + 6-sulfated glucosaminido-alpha-1,4-glucuronic acid oligosaccharide + SO42-
show the reaction diagram
-
heparitinase II
heparitinase II
?
N-acetylated heparan-sulfate
N-acetylated disaccharides
show the reaction diagram
-
heparitinase I
hepartitinase I
?
N-acetylated heparan-sulfate
N-sulfated disaccharides
show the reaction diagram
-
heparitinase I
hepartitinase I
?
N-acetylated, 6-sulfated glucosaminido-alpha-1,4-glucuronic acid
disulfated, N-acetylated, 6-sulfated disaccharides
show the reaction diagram
-
heparitinase II
heparitinase II
?
partially de-N-acetylated polysaccharide of Escherichia coli K5 strain
(DELTA4,5-unsaturated hexuronic acid)-(N-unsubstituted glucosamine)-(hexuronic acid)-(N-acetylglucosamine)
show the reaction diagram
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the polysaccharide consists of the repeating linear sequence -4GlcAbeta1-4GlcNAcalpha1-. Under controlled conditions for partial digestion, lyase III does not act at the GlcN-GlcA linkage, whereas GlcNAc-GlcA is cleaved. Under forced conditions for exhaustive digestion, the GlcN-GlcA linkage is only partially cleaved
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-
?
partially de-N-sulfated forms of heparin
(DELTA4,5-unsaturated hexuronic acid)-(N-unsubstituted glucosamine)
show the reaction diagram
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heparinase III
-
-
?
unsulfated alpha-L-idopyranosyluronic acid
?
show the reaction diagram
-
major requirement for enzyme action is reduced sulfation, cannot cleave linkages containing unsulfated GalAP residues
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-
?
additional information
?
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cleavage/degradation of heparin forming unsaturated uronic acid
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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
heparan sulfate
?
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ba2+
1 mM: 109% activity
Cd2+
-
0.01 mM, stimulates
Cu2+
-
0.01 mM, stimulates
Hg2+
-
0.01 mM, stimulates
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
alpha-lactose
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Cd2+
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1 mM
diethyl dicarbonate
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inactivation, 80% reversible by hydroxylamine within 6 h, mapping of modified histidine residues
DTT
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suppresses the dimerization
Hg2+
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1 mM
LG1PS
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50% inhibition at 470 M
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N-acetylheparin
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-
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Pb2+
1 mM: 0% activity
PEG 8000
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suramin
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50% inhibition at 350 M
Triton X-100
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.009 - 0.191
heparan sulfate
additional information
heparan sulfate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5 - 132
heparan sulfate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
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inactivation kinetics
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1.7
hyaluronic acid as substrate
21.46
heparin as substrate
32.3
heparan sulfate (porcine intestine) as substrate
51.81
heparan sulfate (bovine kidney) as substrate
149
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purified recombinant HepI, pH 7.5, 30C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5
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activity higher at 5.5 than at 4.5
7.5
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assay at
7.6
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22 - 52
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42 - 48
activity above 90%
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.65
calculated for mature protein
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
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propagated in bovine lung microvascular endothelial cells
Manually annotated by BRENDA team
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)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
74930
x * 74930, calculated for mature protein
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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three-dimensional model structure, modelling based on the known crystal structure of Bacteroides thetaiotaomicron HepI, overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
isoform HepIII, to 1.6 A resolution. The overall architecture of HepIII belongs to the (alpha/alpha)5 toroid subclass with an N-terminal toroid-like domain and a C-terminal beta-sandwich domain. An elimination mechanism is suggested whereby Asn260 and His464 neutralize the carboxylic group, whereas Tyr314 serves both as a general base in C-5 proton abstraction, and a general acid in a proton donation to reconstitute the terminal hydroxyl group, respectively
isoform Hep III, to 2.2 A resolution. The enzyme comprises an N-terminal alpha/alpha-barrel domain and a C-terminal antiparallel beta-sheet domain as its basic scaffold. Isoform Hep III exhibits an open form compared with the closed form of Hep II. An active site of Hep III is located in the deep cleft at the interface between its two domains
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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purified recombinant enzyme, half-life is 160 min
39.4
melting temperature, presence of 1 mM heparin tetrasaccharide
40.5
melting temperature, presence of 0.1 mM heparin tetrasaccharide
43.9
melting temperature, presence of 0.1 mM heparin disaccharide
44.3
melting temperature, presence of 0.1 mM N-acetyl glucosamine
45.2
melting temperature, presence of 0.1 mM gellan tetrasaccharide
45.44
melting temperature
70
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purified recombinant enzyme, loss of 97% activity within 1 min
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
after freeze-thawing, 97% of maximal activity remains
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, for at least 2 months
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4C, purified recombinant MBP-HEPI, 1 week, 95% remaining activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
immobilized metal ion affinity chromatography
recombinant His-tagged wild-type and mutants from Escherichia coli
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recombinant maltose-binding protein fusion HepI, MBP-HepI, from Escherichia coli by affinity and anion exchange chromatography, followed by gel filtration to apparent homogeneity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
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expression of maltose-binding protein fusion HepI, MBP-HepI, in Escherichia coli
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expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli, recombinant wild-type enzyme shows a lower activity than the native one
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His-tagged version expressed in Escherichia coli BL21(DE3)
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C352A
87% wild-type activity (heparan sulfate), 72% wild-type activity (heparin)
C474A
32% wild-type activity (heparan sulfate), below 10% wild-type activity (heparin)
C577A
98% wild-type activity (heparan sulfate), 72% wild-type activity (heparin)
C624A
24% wild-type activity (heparan sulfate), below 10% wild-type activity (heparin)
C352A
-
87% wild-type activity (heparan sulfate), 72% wild-type activity (heparin)
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C474A
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32% wild-type activity (heparan sulfate), below 10% wild-type activity (heparin)
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C577A
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98% wild-type activity (heparan sulfate), 72% wild-type activity (heparin)
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C624A
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24% wild-type activity (heparan sulfate), below 10% wild-type activity (heparin)
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C297S
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site-directed mutagenesis, the mutant suppresses the dimerization and shows 70% reduced activity compared to the wild-type enzyme
E237A
40.8% of wild-type activitiy
F423A
42.4% of wild-type activitiy
H105A
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PCR overlap extension site-directed mutagenesis, very low expression level, no measurement of activity possible, reduced expression level
H110A
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PCR overlap extension site-directed mutagenesis, reduced kcat, highly reduced Km compared to both recombinant and native wild-type enzymes, reduced expression level
H139A
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PCR overlap extension site-directed mutagenesis, reduced kcat and increased Km compared to both recombinant and native wild-type enzymes, reduced expression level
H152A
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PCR overlap extension site-directed mutagenesis, reduced Km and a kcat value between the recombinant and the native wild-type enzyme
H225A
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PCR overlap extension site-directed mutagenesis, Km is the same as for the recombinant wild-type, reduced kcat
H234A
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PCR overlap extension site-directed mutagenesis, Km is similar to the recombinant wild-type, reduced kcat
H295A
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PCR overlap extension site-directed mutagenesis, inactive mutant
H36A
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PCR overlap extension site-directed mutagenesis, reduced Km and a kcat value between the recombinant and the native wild-type enzyme
H469A
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PCR overlap extension site-directed mutagenesis, reduced Km and increased kcat compared to both recombinant and native wild-type enzymes
H510A
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PCR overlap extension site-directed mutagenesis, inactive mutant
H539A
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PCR overlap extension site-directed mutagenesis, Km between the recombinant and the native wild-type enzyme, kcat is increased compared to both wild-type enzymes
I29V/L657S
42.7% of wild-type activitiy
N240A
3.1% of wild-type activitiy
Q238A
88.5% of wild-type activitiy
W350A
27.7% of wild-type activitiy
Y294F
2.8% of wild-type activitiy
Y450F
5.6% of wild-type activitiy
Y590
7.7% of wild-type activitiy
E237A
-
40.8% of wild-type activitiy
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F423A
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42.4% of wild-type activitiy
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H241A
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29.7% of wild-type activitiy
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H424A
-
0.3% of wild-type activitiy
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N240A
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3.1% of wild-type activitiy
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Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
temperature-induced reactivation of MBP-HepI, when the temperature is lowered from 35C to 4C, the rate constant of unfolding decreases by 6000times while that of refolding decreases by only 600times, MBP-HepI undergoes reactivation during the cooling treatment at 4C after incubation at 35C
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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analysis of heparitinase induced changes in Xenopus laevis embryos