Information on EC 3.2.1.129 - endo-alpha-sialidase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.2.1.129
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RECOMMENDED NAME
GeneOntology No.
endo-alpha-sialidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
endohydrolysis of (2->8)-alpha-sialosyl linkages in oligo- or poly(sialic) acids
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of O-glycosyl bond
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SYSTEMATIC NAME
IUBMB Comments
polysialoside (2->8)-alpha-sialosylhydrolase
Although the name endo-N-acetylneuraminidase has also been used for this enzyme, this is misleading since its activity is not restricted to acetylated substrates. An exo-alpha-sialidase activity is listed as EC 3.2.1.18 exo-alpha-sialidase. See also EC 4.2.2.15 anhydrosialidase.
CAS REGISTRY NUMBER
COMMENTARY hide
91195-87-8
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
bacteriophage phi1.2
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Manually annotated by BRENDA team
bacteriophage phi92
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UniProt
Manually annotated by BRENDA team
bacteriophage PK1A
bacteriophage PK1E
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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
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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
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
alpha-2,8-glycosidically linked sialic acid + H2O
?
show the reaction diagram
37C
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-
?
alpha-2,8-linked polysialic acid + H2O
?
show the reaction diagram
alpha-2,8-linked polysialic acid + H2O
oligomers of polysialic acid
show the reaction diagram
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-
-
-
?
alpha-2,8-linked polysialic acid cross-linked by diepoxyoctane + H2O
oligomers of polysialic acid
show the reaction diagram
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polysialic acid hydrogel consisting of 20% (w/v) polysialic acid and a minimum of 0.6 equivalents diepoxyoctane, pH 7.4, 37°C
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?
alpha-2,8-linked sialic acid residues cross-linked by diepoxyoctane + H2O
?
show the reaction diagram
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hydrogel with minimum chain length of 8
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-
?
capsular polysaccharides + H2O
?
show the reaction diagram
bacteriophage phi92
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-
-
?
K1 antigen
?
show the reaction diagram
longchain alpha 2,8-linked polysialic acid + H2O
oligomers of alpha 2,8-linked polysialic acid
show the reaction diagram
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minimum substrate is tetrameric polysialic acid, processive enzyme activity on oligomers larger than that, confirmation by H NMR spectroscopy and anion-exchange chromatography
major product is an oligomer consisting of 3 monomers in wild-type and cleavage mutant S911A, in binding site mutant R837A/S848A more random oligomers are produced
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?
poly(sialic) acid + H2O
?
show the reaction diagram
poly(sialic) acid + H2O
fragments of poly(sialic) acid
show the reaction diagram
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-
-
-
?
poly(sialic) acids or oligo(sialic) acids containing alpha-2,8-linked N-acetylneuraminic acid + H2O
fragments of poly(sialic) acid or oligo(sialic) acids
show the reaction diagram
polysialic acid capsules of bacteria + H2O
oligomers of alpha 2,8-linked polysialic acid
show the reaction diagram
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minimum substrate is tetrameric polysialic acid, processive enzyme activity on oligomers larger than that, confirmation by H NMR spectroscopy and anion-exchange chromatography
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?
polysialylated neural cell adhesion molecule + H2O
?
show the reaction diagram
trifluoromethylumbelliferyl sialotetraoside + H2O
trifluoromethylumbelliferone + sialotetraoside
show the reaction diagram
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?
trifluoromethylumbelliferyl sialotrioside + H2O
trifluoromethylumbelliferone + sialotrioside
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
alpha-2,8-linked polysialic acid + H2O
?
show the reaction diagram
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specifically cleaves alpha 2,8-linked sialic acid residues with minimum chain length of 8, no effect on other sialic acid containing structures
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?
alpha-2,8-linked polysialic acid + H2O
oligomers of polysialic acid
show the reaction diagram
-
-
-
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?
capsular polysaccharides + H2O
?
show the reaction diagram
bacteriophage phi92
I7HXG2
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?
poly(sialic) acids or oligo(sialic) acids containing alpha-2,8-linked N-acetylneuraminic acid + H2O
fragments of poly(sialic) acid or oligo(sialic) acids
show the reaction diagram
polysialic acid capsules of bacteria + H2O
oligomers of alpha 2,8-linked polysialic acid
show the reaction diagram
-
minimum substrate is tetrameric polysialic acid, processive enzyme activity on oligomers larger than that, confirmation by H NMR spectroscopy and anion-exchange chromatography
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?
polysialylated neural cell adhesion molecule + H2O
?
show the reaction diagram
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ca2+
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
7.41 - 10.6
K1 antigen
1.2 - 1.6
oligosialic acid
0.0066 - 27.1
polysialic acid
0.85
tetrameric silica acid
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identical kinetic parameters for wild type and cleavage site mutant S911A
0.68
trifluoromethylumbelliferyl sialotrioside
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at pH 4.5
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.01 - 1.5
polysialic acid
4.37
tetrameric silica acid
Enterobacteria phage K1F
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identical kinetic parameters for wild type and cleavage site mutant S911A
1.28
trifluoromethylumbelliferyl sialotrioside
Enterobacteria phage K1F
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at pH 4.5
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.95
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after DEAE-Sephadex chromatography
additional information
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intravitreal endo-N injection and axotomy of optical nerve in wild-type mice: 4 days after treatment no difference in retinal ganglion cell density compared with untreated/uninjured, and vehicle treated/axotomy controls, at day 7 50% reduced density in vehicle treated/axotomy group compared to untreated/uninjured group, an additional 27% density reduction in endo-N treatment group; intravitreal vehicle injection and axotomy of optical nerve in NCAM-/-mice: 4 days after treatment reduced density in vehicle injection/axotomy group compared to untreated/uninjured group, no neuroprotective effect of injection; no differences in retinal ganglion cell densities after 14 days in NCAM-/-mice with or without endo-N treatment, shows non-toxicity of endo-N treatment itself; polysialic acid is degraded from inner retina in vivo within 6 hours by intravitreal injection of 1 microl (6.7 U/microl) endo-N in 50% phosphate buffered saline with glycerol, from the outer retina within 24 hours, and remains absent at day 14l; polysialic acid is degraded in vitro (neonatal cell culture) within 1 day by 1 microl and 3 microl endo-N, not by 0.5 microl (6.7 U/microl) in 50% phosphate buffered saline with glycerol; polysialylated neural cell adhesion molecules are degraded in vitro (neonatal cell culture) within 12 hours after treatment with 1 microl (6.7 U/microl) in 50% phosphate buffered saline with glycerol, and remains absent 5 days after treatment, fewer retinal ganglion cells (54%) than control or vehicle control (96%); treatment with 1 microl (6.7 U/microl) in 50% phosphate buffered saline with glycerol, no significant difference in retinal ganglion cell densities 7 days after treatment, at day 14 reduction by approximately one third compared with control and vehicle control
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.2 - 5.5
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
89000
sequence analysis
119000
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deduced from nucleotide sequence
208000
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enzyme complex, SDS-PAGE, solubilized in SDS-mercaptoethanol-containing sample buffer at 37C
210000
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gel filtration
328000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homotrimer
trimer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
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proteolysis of the C-terminal domain that functions as an intramolecular chaperone and is released during enzyme maturation of wild-type enzyme
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in complex with a dimer of alpha-2,9-linked sialic acid, hanging drop vapor diffusion method, using 6% (w/v) PEG3350, 70 mM CHES buffer, pH 9.5 and 70 mM SrCl2
bacteriophage phi92
crystal structure of the catalytic domain of endoN from caliphate K1F reveals a functional trimer, folding is mediated by an intramolecular C-terminal chaperone domain
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hanging drop vapor diffusion method, using 16% (w/v) PEG 8000, 0.1 M Tris-HCl pH 7.2, 3% (v/v) 2-propanol
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in complex with oligomeric sialic acid, 1.9 A resolution
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wild type endoNF cocrystallized with oligomeric sialic acid and mutant enzymes H350A and R647A are crystallized by the hanging drop vapor diffusion method, using
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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the enzyme displays outstanding stability and resistance to SDS
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
amylose resin column chromatography, gel filtration
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by gel filtration, affinity chromatography and a Ni2+-chelating column
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description in Schwarzer, D. et al. (2007) J.Biol. Chem. 282, 2821-2831
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HiTrap Q HP anion exchange column chromatography and Superdex 200 gel filtration
bacteriophage phi92
Ni affinity column chromatography
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recombinant enzyme
recombinant protein from Escherichia coli
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; expression in Escherichia coli as fusion protein
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expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli BL21-Gold(DE3) cells
bacteriophage phi92
expression in Escherichia coli
expression in Escherichia coli as fusion protein
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expression in Escherichia coli BL21(DE3)
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mutant proteins harbouring single amino acid substitutions expressed as GFP-fusion proteins
protein expression in Escherichia coli BL21-Gold(DE3) in the presence of 100 microgram/ml Carbenicillin, 30°C
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subcloned into the NdeI and XhoI restriction sites of pET22b, resulting in a construct encoding the C-terminal domain of endoNF with a C-terminal His6 tag, expressed in Escherichia coli BL21-Gold(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D489N
back-mutations, partial reduction of enzymic activity. Single amino acid substitution do not inactivate the catalytic activity totally
H332N/N489D
complete inactivation
H417Y/N489D
complete inactivation
R614G
displays full endosialidase activity
Y417H
back-mutations, partial reduction of enzymic activity. Single amino acid substitution do not inactivate the catalytic activity totally
D138A
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lower Km than wild-type enzyme
D154A
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lower Km than wild-type enzyme
DELTA DI
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insoluble enzyme
DELTA DII
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insoluble enzyme
DELTA P-loop
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higher Km than wild-type enzyme
G153A
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less turnover than wild-type enzyme
R614G
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displays full endosialidase activity
Trunc N45
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lower Km than wild-type enzyme
W159A
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insoluble enzyme
G956A
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completely loses enzymatic activity
H350A
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1.2% relative activity compared to the wild type enzyme
H350N
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3.4% relative activity compared to the wild type enzyme
H350Q
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4.4% relative activity compared to the wild type enzyme
H542A
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36% relative activity compared to the wild type enzyme
H954A
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completely loses enzymatic activity
K410A
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20% relative activity compared to the wild type enzyme
N912A
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completely loses enzymatic activity
Q853A
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binding site mutation, active within control range with soluble polysialic acid
R1035A
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completely loses enzymatic activity
R547A/E581A
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inactive, but no effect on expression, maturation or comlex formation
R549A
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0.6% relative activity compared to the wild type enzyme
R596A
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behaves like wild-type in terms of expression level, maturation by proteolytic cleavage and trimer formation
R596A/E581A
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inactive, but no effect on expression, maturation or comlex formation
R596A/R647A
R596A/R647A/Q853A
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active site mutation plus binding site mutation, EC50: 5.0 nM surface bound polysialic acid
R596A/R647A/R837A
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active site mutation plus binding site mutation, 5-fold increased EC50: 11 nM polysialic acid
R596A/R647A/R837A/S848A
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active site mutation plus binding site mutation, EC50: 30 nM surface bound polysialic acid, increased EC50: 30 nM polysialic acid
R596A/R647A/S848A
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active site mutation plus binding site mutation, only binding site mutant with SDS resistance which is a criterion for kinetic stabilization of the enzyme, EC50: 4.1 nM surface bound polysialic acid
R596A/R647A/S848A/Q853A
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active site mutation plus binding site mutation, increased EC50: 6.2 nM polysialic acid
R596A/R647A/S911A
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active site mutation plus cleavage site mutation, tremendously increased EC50: 360 nM polysialic acid
R837A
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binding site mutation, increased molar activity with soluble polysialic acid
R837A/Q853A
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binding site mutation, insoluble enzyme
R837A/S848A
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binding site mutation, increased molar activity with soluble polysialic acid
R837A/S848A/Q853A
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binding site mutation, insoluble enzyme
S848A
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binding site mutation, active within control range with soluble polysialic acid
S848A/Q853A
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binding site mutation, active within control range with soluble polysialic acid
W328R
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6.0% relative activity compared to the wild type enzyme
R596A
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exhibits a more than 95% decreased activity compared to wild-type endoNF, but binds polysialic acid
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
degradation
medicine
molecular biology
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removal of target molecules to investigate their function
additional information