Information on EC 3.2.1.129 - endo-alpha-sialidase

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The expected taxonomic range for this enzyme is: Viruses

EC NUMBER
COMMENTARY
3.2.1.129
-
RECOMMENDED NAME
GeneOntology No.
endo-alpha-sialidase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
endohydrolysis of (2->8)-alpha-sialosyl linkages in oligo- or poly(sialic) acids
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
hydrolysis of O-glycosyl bond
-
-
-
-
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.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
alpha-2,8-sialosylhydrolase
-
-
-
-
endo-N-acetylneuraminidase
-
-
-
-
endo-N-acetylneuraminidase
A4UUQ0
-
endo-N-acetylneuraminidase
-
-
endo-N-acetylneuraminidase
-
-
endo-sialidase
-
-
endoE
Escherichia coli A192PP
-
-
-
endoneuraminidase
-
-
-
-
endoneuraminidase
-
-
endoNF
-
endoNF is an inverting sialidase
endoNF
-
from coliphage K1F
endoNF
-
from coliphage K1F, lacking N-terminal capsid binding domain
endoNF
-
proteolyitcally processed endosialidase lacking the N-terminal head-binding domain
endosialidase
-
-
-
-
endosialidase
A4UUQ0
-
endosialidase
-
-
endosialidase
-
-
endosialidase E
-
-
endosialidase E
Escherichia coli A192PP
-
-
-
endosialidase NF
-
-
endosialidase NF
Q04830
-
endosialidase NF
-
-
G102
-
-
-
-
neuraminidase, endo-
-
-
-
-
poly(alpha-2,8-sialoside) alpha-2,8-sialosylhydrolase
-
-
-
-
poly(alpha-2,8-sialosyl) endo-N-acetylneuraminidase
-
-
-
-
poly(sialoside) alpha-2,8-sialosylhydrolase
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
91195-87-8
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
bacteriophage phi1.2
-
-
-
Manually annotated by BRENDA team
bacteriophage PK1A
-
-
-
Manually annotated by BRENDA team
bacteriophage PK1A
host strain: Escherichia coli K1 (IH3088)
-
-
Manually annotated by BRENDA team
bacteriophage PK1E
-
-
-
Manually annotated by BRENDA team
bacteriophage PK1E
host strain: Escherichia coli K1 (IH3088)
-
-
Manually annotated by BRENDA team
enzyme lacking the N-terminal head-binding domain
-
-
Manually annotated by BRENDA team
Escherichia coli A192PP
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
physiological function
-
recombinant capsule depolymerase, endosialidase E, selectively removes polysialic acid from the bacterial surface
physiological function
Escherichia coli A192PP
-
recombinant capsule depolymerase, endosialidase E, selectively removes polysialic acid from the bacterial surface
-
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
alpha-2,8-glycosidically linked sialic acid + H2O
?
show the reaction diagram
-, Q04830
37C
-
-
?
alpha-2,8-linked polysialic acid + H2O
?
show the reaction diagram
-
-
-
-
?
alpha-2,8-linked polysialic acid + H2O
?
show the reaction diagram
-
specifically cleaves alpha 2,8-linked sialic acid residues with minimum chain length of 8, no effect on other sialic acid containing structures
-
-
?
alpha-2,8-linked polysialic acid + H2O
oligomers of polysialic acid
show the reaction diagram
-
-
-
-
?
alpha-2,8-linked polysialic acid cross-linked by diepoxyoctane + H2O
oligomers of polysialic acid
show the reaction diagram
-
polysialic acid hydrogel consisting of 20% (w/v) polysialic acid and a minimum of 0.6 equivalents diepoxyoctane, pH 7.4, 37°C
-
-
?
alpha-2,8-linked sialic acid residues cross-linked by diepoxyoctane + H2O
?
show the reaction diagram
-
hydrogel with minimum chain length of 8
-
-
?
K1 antigen
?
show the reaction diagram
-
-
-
-
?
K1 antigen
?
show the reaction diagram
-
-
-
-
?
poly(sialic) acid + H2O
?
show the reaction diagram
-
-
-
-
?
poly(sialic) acid + H2O
?
show the reaction diagram
-
-
-
-
?
poly(sialic) acid + H2O
?
show the reaction diagram
A4UUQ0
-
-
-
?
poly(sialic) acid + H2O
?
show the reaction diagram
-
all sialic acid residues (Sia1-5) are bound by endoNF
-
-
?
poly(sialic) acid + H2O
fragments of poly(sialic) acid
show the reaction diagram
-
-
-
-
?
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
-
-
-
-
?
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
-
-
-
?
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
-
-
-
-
?
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
bacteriophage phi1.2
-
-
-
?
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
bacteriophage PK1A
-
-
-
?
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
bacteriophage PK1A
-
-
-
-
?
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
bacteriophage PK1E
-
-
-
?
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
bacteriophage PK1E
-
-
-
?
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
-
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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 capsule isolated from Escherichia coli N67 or Escherichia coli K1, the minimum requirement for cleavage is alpha-2,8-(NeuAc)5, bacterial and neural membrane glycoconjugates
-
?
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
-
no release of alpha-2,3-, alpha-2,6-, or alpha-2,9-linked sialic residues from a variety of other substrates
-
?
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
-
oligosialic acid from Escherichia coli K1
-
?
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
-
brain glycoproteins
-
?
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
bacteriophage PK1A
-
brain glycoproteins
-
?
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
-
catalyzes the hydrolysis of both alpha-2,8-linked poly(N-acetyl-D-neuraminic acid) and poly(N-glycoloyl-D-neuraminic acid)
-
?
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
-
limited hydrolysis of the Escherichia coli K92 antigen, an N-acetylneuraminic acid homopolymer containing alternating alpha-2,8-, and alpha-2,9-linkages
-
?
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
bacteriophage PK1A
-
enzyme requires the simultaneous presence adjacent to the site of cleavage a minimum of 3 sialic acid residues on the distal side and a minimum of 5 sialic acid residues on the proximal side
-
?
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
-
rapid hydrolysis of both the O-acetylated and non-O-acetylted forms of the K1 antigen, an alpha-2,8-linked homopolymer of N-acetylneuraminic acid, and of Meningococcus B antigen
-
?
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
-
-
?
polysialylated neural cell adhesion molecule + H2O
?
show the reaction diagram
-
-
-
-
?
polysialylated neural cell adhesion molecule + H2O
?
show the reaction diagram
-
polysialic acid is an unbranched homopolymer of 100-300 alpha-2,8-linked silica acid residues
-
-
?
trifluoromethylumbelliferyl sialotetraoside + H2O
trifluoromethylumbelliferone + sialotetraoside
show the reaction diagram
-
-
-
-
?
trifluoromethylumbelliferyl sialotrioside + H2O
trifluoromethylumbelliferone + sialotrioside
show the reaction diagram
-
-
-
-
?
longchain alpha 2,8-linked polysialic acid + 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
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
-
?
additional information
?
-
-
the endo-sialidase requires the occupation of a minimum of three subsites by sialic acid for efficient catalysis, so neither monomer (trifluoromethylumbelliferyl sialoside) nor dimer (trifluoromethylumbelliferyl sialobioside) are hydrolyzed by endoNF
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
alpha-2,8-linked polysialic acid + H2O
?
show the reaction diagram
-
specifically cleaves alpha 2,8-linked sialic acid residues with minimum chain length of 8, no effect on other sialic acid containing structures
-
-
?
alpha-2,8-linked polysialic acid + H2O
oligomers of polysialic acid
show the reaction diagram
-
-
-
-
?
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
-
-
-
-
?
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
bacteriophage phi1.2
-
-
-
-
?
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
bacteriophage PK1A
-
-
-
-
?
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
bacteriophage PK1E
-
-
-
-
?
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
-
-
-
-
?
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
-
-
?
polysialylated neural cell adhesion molecule + H2O
?
show the reaction diagram
-
-
-
-
?
polysialylated neural cell adhesion molecule + H2O
?
show the reaction diagram
-
polysialic acid is an unbranched homopolymer of 100-300 alpha-2,8-linked silica acid residues
-
-
?
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
7.41
-
K1 antigen
-
37C, pH 6.5, an alpha-2,8-linked homopolymer of N-acetylneuraminic acid
-
10.6
-
K1 antigen
-
37C, pH 6.5
-
1.2
-
oligosialic acid
-
pH 7.5, isolated from Escherichia coli K1, phage-bound enzyme
-
1.6
-
oligosialic acid
-
pH 7.5, from Escherichia coli K1, soluble enzyme
-
0.0066
-
polysialic acid
-
pH 7.5, capsule isolated from Escherichia coli N67, soluble enzyme
0.051
-
polysialic acid
-
pH 7.5, capsule isolated from Escherichia coli K1, soluble enzyme
0.071
-
polysialic acid
-
pH 7.5, capsule isolated from Escherichia coli K1, phage-bound enzyme
1
17.7
polysialic acid
-
37C, pH 6.5, G153A
3.7
-
polysialic acid
-
37C, pH 6.5, Trunc N45
4.2
17.7
polysialic acid
-
37C, pH 6.5, wild-type
6.4
-
polysialic acid
-
37C, pH 6.5, D154A
8.3
-
polysialic acid
-
37C, pH 6.5, D138A
27.1
-
polysialic acid
-
37C, pH 6.5, DELTA P-loop
0.85
-
tetrameric silica acid
-
identical kinetic parameters for wild type and cleavage site mutant S911A
0.68
-
trifluoromethylumbelliferyl sialotrioside
-
at pH 4.5
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.01
1
polysialic acid
-
37C, pH 6.5, G153A
0.2
-
polysialic acid
-
37C, pH 6.5, D138A
0.4
1.5
polysialic acid
-
37C, pH 6.5, wild-type
0.5
-
polysialic acid
-
37C, pH 6.5, Trunc N45
1.2
-
polysialic acid
-
37C, pH 6.5, D154A
1.4
-
polysialic acid
-
37C, pH 6.5, DELTA P-loop
4.37
-
tetrameric silica acid
-
identical kinetic parameters for wild type and cleavage site mutant S911A
1.28
-
trifluoromethylumbelliferyl sialotrioside
-
at pH 4.5
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
0.95
-
-
after DEAE-Sephadex chromatography
additional information
-
-
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
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5.2
5.5
-
-
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
additional information
-
neonatal rat Schwann cell culture (1-3 days postnatal), adult rat Schwann cell culture from 3 months old females, neural stem cells of fetal rat cell culture (gestational day 12), dorsal ganglionic cells of neonatal rat cell culture (2-3 days postnatal)
Manually annotated by BRENDA team
additional information
-
density of mouse retinal ganglion cells after removal of polysialic acid by endo-N treatment to investigate possible toxic effects of endo-N treatment; retinal ganglion cell cultures prepared from Long-Evans rat litters at postnatal day 7-8, and from 10-15 weeks old rats, for in vitro experiments
Manually annotated by BRENDA team
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
76000
-
A4UUQ0
-
89000
-
A4UUQ0
sequence analysis
119000
-
-
deduced from nucleotide sequence
208000
-
-
enzyme complex, SDS-PAGE, solubilized in SDS-mercaptoethanol-containing sample buffer at 37C
210000
-
-
gel filtration
328000
-
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 74000 + x * 38500, SDS-PAGE with boiled samples at 100C
?
-
x * 74000, SDS-PAGE
?
-
x * 76000, SDS-PAGE
?
bacteriophage PK1E
-
x * 74000, SDS-PAGE
homotrimer
-
x-ray crystallography
monomer
A4UUQ0
-
trimer
-
3 * 105000, SDS-PAGE
trimer
-
3 * 103000, SDS-PAGE with boiled samples
trimer
-
crystal structure
trimer
-
gel filtration
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
proteolytic modification
-
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 ACCESSION NO.
LITERATURE
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
-
hanging drop vapor diffusion method, using 16% (w/v) PEG 8000, 0.1 M Tris-HCl pH 7.2, 3% (v/v) 2-propanol
-
in complex with oligomeric sialic acid, 1.9 A resolution
-
wild type endoNF cocrystallized with oligomeric sialic acid and mutant enzymes H350A and R647A are crystallized by the hanging drop vapor diffusion method, using
-
ORGANIC SOLVENT
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
the enzyme displays outstanding stability and resistance to SDS
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
-
bacteriophage PK1E
-
recombinant enzyme
-
amylose resin column chromatography, gel filtration
-
by gel filtration, affinity chromatography and a Ni2+-chelating column
-
description in Schwarzer, D. et al. (2007) J.Biol. Chem. 282, 2821-2831
-
recombinant enzyme
-
recombinant protein from Escherichia coli
-
Ni affinity column chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
mutant proteins harbouring single amino acid substitutions expressed as GFP-fusion proteins
A4UUQ0
; expression in Escherichia coli as fusion protein
-
expression in Escherichia coli
-
expression in Escherichia coli as fusion protein
-
expressed in Escherichia coli BL21(DE3) cells
-
expression in Escherichia coli
-
expression in Escherichia coli BL21(DE3)
-
protein expression in Escherichia coli BL21-Gold(DE3) in the presence of 100 microgram/ml Carbenicillin, 30°C
-
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)
-
expressed in Escherichia coli BL21(DE3) cells
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
D489N
A4UUQ0
back-mutations, partial reduction of enzymic activity. Single amino acid substitution do not inactivate the catalytic activity totally
H332N/N489D
A4UUQ0
complete inactivation
R614G
A4UUQ0
displays full endosialidase activity
Y417H
A4UUQ0
back-mutations, partial reduction of enzymic activity. Single amino acid substitution do not inactivate the catalytic activity totally
D138A
-
lower Km than wild-type enzyme
D154A
-
lower Km than wild-type enzyme
DELTA DI
-
insoluble enzyme
DELTA DII
-
insoluble enzyme
DELTA P-loop
-
higher Km than wild-type enzyme
R614G
-
displays full endosialidase activity
Trunc N45
-
lower Km than wild-type enzyme
W159A
-
insoluble enzyme
E581A
-
behaves like wild-type in terms of expression level, maturation by proteolytic cleavage and trimer formation
E581A
-
the mutation results in complete loss of sialidase activity
E581A
-
1.8% relative activity compared to the wild type enzyme
G956A
-
completely loses enzymatic activity
H350A
-
1.2% relative activity compared to the wild type enzyme
H350N
-
3.4% relative activity compared to the wild type enzyme
H350Q
-
4.4% relative activity compared to the wild type enzyme
H542A
-
36% relative activity compared to the wild type enzyme
H954A
-
completely loses enzymatic activity
N912A
-
completely loses enzymatic activity
Q853A
-
binding site mutation, active within control range with soluble polysialic acid
R1035A
-
completely loses enzymatic activity
R547A/E581A
-
inactive, but no effect on expression, maturation or comlex formation
R549A
-
0.6% relative activity compared to the wild type enzyme
R596A
-
behaves like wild-type in terms of expression level, maturation by proteolytic cleavage and trimer formation
R596A/E581A
-
inactive, but no effect on expression, maturation or comlex formation
R596A/R647A
-
inactive, but no effect on expression, maturation or comlex formation
R596A/R647A
-
control active site mutation without affecting maturation or binding, EC50: 1.9 nM polysialic acid
R596A/R647A/Q853A
-
active site mutation plus binding site mutation, EC50: 5.0 nM surface bound polysialic acid
R596A/R647A/R837A
-
active site mutation plus binding site mutation, 5-fold increased EC50: 11 nM polysialic acid
R596A/R647A/R837A/S848A
-
active site mutation plus binding site mutation, EC50: 30 nM surface bound polysialic acid, increased EC50: 30 nM polysialic acid
R596A/R647A/S848A
-
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
-
active site mutation plus binding site mutation, increased EC50: 6.2 nM polysialic acid
R596A/R647A/S911A
-
active site mutation plus cleavage site mutation, tremendously increased EC50: 360 nM polysialic acid
R647A
-
behaves like wild-type in terms of expression level, maturation by proteolytic cleavage and trimer formation
R647A
-
0.17% relative activity compared to the wild type enzyme
R837A
-
binding site mutation, increased molar activity with soluble polysialic acid
R837A/Q853A
-
binding site mutation, insoluble enzyme
R837A/S848A
-
binding site mutation, increased molar activity with soluble polysialic acid
R837A/S848A/Q853A
-
binding site mutation, insoluble enzyme
S848A
-
binding site mutation, active within control range with soluble polysialic acid
S848A/Q853A
-
binding site mutation, active within control range with soluble polysialic acid
S911A
-
mutant deltaN-endoNF lacking the capsid binding domain but retaining the C-terminal domain, prevents cleavage but not assembly into active trimers
S911A
-
cleavage site mutation: prevents proteolysis of the C-terminal domain that functions as an intramolecular chaperone and is normally released during enzyme maturation. Substrate binding is reduced similarly to binding site mutations. Altered activities: 3times higher activity with soluble polycialic acid as substrate, 190fold reduced activity with immobilized polysialic acid as substrate, no difference in activity with minimal substrate tetrameric sialic acid
W328R
-
6.0% relative activity compared to the wild type enzyme
R596A
-
exhibits a more than 95% decreased activity compared to wild-type endoNF, but binds polysialic acid
H417Y/N489D
A4UUQ0
complete inactivation
additional information
A4UUQ0
N-terminal deletion mutants are catalytically active, whereas all C-terminally truncated mutants are inactive
G153A
-
less turnover than wild-type enzyme
additional information
-
point mutation located on or near the putative active site, exhibits greatly reduced activity
K410A
-
20% relative activity compared to the wild type enzyme
additional information
-
mutant deltaN-endoNF lacking the capsid binding domain, forms trimeric complexes
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
biotechnology
-
degradation of non-toxic modified polysialic acid hydrogel scaffold in neuro-regenerative tissue engineering (4.26 microgram enzyme + 39 mg hydrogel), in phosphate buffered saline (400 microl, pH 7.4), at 37°C, degradation speed 2-11 days depending on cross-linker amount (0.6, 0.8, 2 equivalents diepoxyoctane, no activity with 3 equivalents diepoxyoctane), hydrogel was coated with collagen I, poly-L-lysine/collagen I, or diluted matrigel for neurite formation in PC12 cells
biotechnology
-
degradation of non-toxic modified polysialic acid hydrogel scaffold in neuro-regenerative tissue engineering: no degradation in 12 days with 1 microg/ml active enzyme + 105 cubic mm hydrogel in phosphate buffered saline (pH 7.4), at room temperature, increase to 4 microg/ml at end of week 2 initiates degradation, total degradation after 4 weeks, hydrogel was coated with poly-L-lysine, poly-L-ornithine-laminin or collagen for neurite formation in neonatal and adult rat Schwann cells, neural rat stem cells, and dorsal root ganglionic cells from rats
degradation
-
degradation of non-toxic modified polysialic acid hydrogel scaffold in neuro-regenerative tissue engineering (4.26 microgram enzyme + 39 mg hydrogel), in phosphate buffered saline (400 microl, pH 7.4), at 37°C, degradation speed 2-11 days depending on cross-linker amount (0.6, 0.8, 2 equivalents diepoxyoctane, no activity with 3 equivalents diepoxyoctane), hydrogel was coated with collagen I, poly-L-lysine/collagen I, or diluted matrigel for neurite formation in PC12 cells
degradation
-
degradation of non-toxic modified polysialic acid hydrogel scaffold in neuro-regenerative tissue engineering: no degradation in 12 days with 1 microg/ml active enzyme + 105 cubic mm hydrogel in phosphate buffered saline (pH 7.4), at room temperature, increase to 4 microg/ml at end of week 2 initiates degradation, total degradation after 4 weeks, hydrogel was coated with poly-L-lysine, poly-L-ornithine-laminin or collagen for neurite formation in neonatal and adult rat Schwann cells, neural rat stem cells, and dorsal root ganglionic cells from rats
medicine
-
degradation of non-toxic modified polysialic acid hydrogel scaffold in neuro-regenerative tissue engineering (4.26 microgram enzyme + 39 mg hydrogel), in phosphate buffered saline (400 microl, pH 7.4), at 37°C, degradation speed 2-11 days depending on cross-linker amount (0.6, 0.8, 2 equivalents diepoxyoctane, no activity with 3 equivalents diepoxyoctane), hydrogel was coated with collagen I, poly-L-lysine/collagen I, or diluted matrigel for neurite formation in PC12 cells
medicine
-
degradation of non-toxic modified polysialic acid hydrogel scaffold in neuro-regenerative tissue engineering: no degradation in 12 days with 1 microg/ml active enzyme + 105 cubic mm hydrogel in phosphate buffered saline (pH 7.4), at room temperature, increase to 4 microg/ml at end of week 2 initiates degradation, total degradation after 4 weeks, hydrogel was coated with poly-L-lysine, poly-L-ornithine-laminin or collagen for neurite formation in neonatal and adult rat Schwann cells, neural rat stem cells, and dorsal root ganglionic cells from rats
medicine
-
investigation of possible role of polysialic neural cell adhesion molecules in the pathophysiology of epilepsy
molecular biology
-
removal of target molecules to investigate their function
additional information
-
the C-terminal domain plays a crucial role in folding and assembling not only the C-terminal domain of endosialidases but also of other, unrelated phage proteins
additional information
-
almost complete removal of polysialic acid by endo-N injection into the brain 5 days after stimulation (return to normal polysialic acid levels after 11 weeks): no effect on proliferation, neurogenesis, and the fate of newborn cells in the hippocampus of rats without status epileptics. Increase of newborn cells in status epileptics rats compared to controls without significant difference between endo-N and vehicle treatment. However, endo-N treatment reduces the total number of newborn neurons (64%) upon induction of the status epileptics compared to vehicle treatment. No endo-N effect on hilar basal dendrite generation compared to vehicle control. No effect on doublecortin-expressing neuronal progenitor cells, and no effect on subpopulation of these cells with persistent basal dendrites compared to vehicle control. No difference in number, severity and duration of seizures between groups. Spatial learning deficit of status epilepticus mice is reduced by end-N treatment compared to vehicle control