Information on EC 3.5.1.52 - peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase

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

EC NUMBER
COMMENTARY hide
3.5.1.52
-
RECOMMENDED NAME
GeneOntology No.
peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Hydrolysis of an N4-(acetyl-beta-D-glucosaminyl)asparagine residue in which the glucosamine residue may be further glycosylated, to yield a (substituted) N-acetyl-beta-D-glucosaminylamine and a peptide containing an aspartate residue
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis
hydrolysis of carboxylic acid amide
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
N-linked-glycopeptide-(N-acetyl-beta-D-glucosaminyl)-L-asparagine amidohydrolase
Does not act on (GlcNAc)Asn, because it requires the presence of more than two amino-acid residues in the substrate [cf. EC 3.5.1.26, N4-(beta-N-acetylglucosaminyl)-L-asparaginase]. The plant enzyme was previously erroneously listed as EC 3.2.2.18.
CAS REGISTRY NUMBER
COMMENTARY hide
83534-39-8
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
fungus
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
gene Ddpngase
UniProt
Manually annotated by BRENDA team
gene Ddpngase
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
rice
-
-
Manually annotated by BRENDA team
medaka fish
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Prunus amygdalus var. dulcis
almond
-
-
Manually annotated by BRENDA team
white campion
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
acetylcholine receptor + H2O
?
show the reaction diagram
-
-
-
-
?
alpha-dansyl derivative fetuin pentaglycopeptide + H2O
?
show the reaction diagram
alpha1-antitrypsin Pi Z + H2O
deglycosylated alpha1-antitrypsin Pi Z
show the reaction diagram
-
-
-
?
Antithrombin III + H2O
?
show the reaction diagram
-
significant amount of only partly de-N-glycosylated protein is detected even after more than 18 h incubation time with PNGase F
-
-
?
asialoglycopeptide I + H2O
?
show the reaction diagram
-
fetuin-derived radio-labeled substrate, deglycosylation
-
-
?
ATPase p97 + H2O
?
show the reaction diagram
bence-jones wh lambda glycopeptide + H2O
?
show the reaction diagram
-
acid PNGase M from blastoderm stage 11
-
-
?
beta-aspartylglycosylamine + H2O
aspartic acid + NH3 + N-acetylglucosamine
show the reaction diagram
bovine fetuin glycopeptide + H2O
?
show the reaction diagram
class I MHC HC + H2O
?
show the reaction diagram
coagulation factor IX + H2O
?
show the reaction diagram
-
almost quantitative de-N-glycosylation with PNGase F is observed after a rather short time
-
-
?
concanavalin A-precursor + H2O
?
show the reaction diagram
-
deglycosylation leads to conversion into an active lectin
-
-
?
Cys-Gly-Leu-Val-Pro-Val-Leu-Ala-Glu-Asn-Tyr-Asn(Man3Gal2GlcNAc4NeuAc2)-Lys + H2O
?
show the reaction diagram
-
-
-
?
dabsyl-Gly-Glu-Asn-(GlcNAc4Man3)-Arg + H2O
?
show the reaction diagram
dEDANSylated hen ovomucoid glycopeptide + H2O
?
show the reaction diagram
denatured ribonuclease B + H2O
?
show the reaction diagram
-
-
-
-
?
denatured RNase B + H2O
?
show the reaction diagram
-
SpPNGase cleaves N-glycan from denatured RNase B
-
-
?
denatured RNaseB + H2O
deglycosylated RNaseB
show the reaction diagram
-
-
-
?
fetuin glycopeptide II + H2O
?
show the reaction diagram
-
acid PNGase M from blastoderm stage 11
-
-
?
fetuin-derived asialoglycopeptide I + H2O
?
show the reaction diagram
-
([14C]-CH3)2Leu-Asn(GlcNAc5-Man3Gal3)-Asp-Ser-Arg
-
-
?
Gal2GlcNAc2Man3GlcNAc2-Asn-peptide + H2O
?
show the reaction diagram
-
-
-
-
?
GlcNAc-Asn-peptide + H2O
?
show the reaction diagram
-
-
-
-
?
glycopeptide + H2O
?
show the reaction diagram
glycoprotein US11 + H2O
?
show the reaction diagram
-
the enzyme deglycolyzes HCMV glycoprotein US11
-
-
?
glycoprotein US2 + H2O
?
show the reaction diagram
-
the enzyme deglycolyzes HCMV glycoprotein US2
-
-
?
HR23-ubiquitin-like domain + H2O
?
show the reaction diagram
-
the N-terminal domain of PNGase (PUB) interacts with HR23-ubiquitin-like domain and ubiquitin chains
-
-
?
human asialotransferrin + H2O
?
show the reaction diagram
-
-
-
-
?
human IgG + H2O
?
show the reaction diagram
-
-
-
-
?
human transferrin glycopeptide + H2O
?
show the reaction diagram
-
-
-
?
L-hyosophorin + H2O
?
show the reaction diagram
-
neutral and acid PNGase M
-
-
?
laccase + H2O
?
show the reaction diagram
-
-
-
-
?
Leu-Asn(GlcNAc5Man3Gal3)-Asp-Ser-Arg + H2O
?
show the reaction diagram
fetuin-derived asialoglycopeptide I, 14C, substrate activity assay
-
-
?
Man5GlcNAc2-Asn-peptide + H2O
?
show the reaction diagram
-
-
-
-
?
melanopsin + H2O
?
show the reaction diagram
-
-
-
?
mu opioid receptor + H2O
?
show the reaction diagram
-
the enzyme removes all N-linked glycans
-
-
?
N-glycoprotein + H2O
?
show the reaction diagram
-
PNGase is involved in the release of N-glycans from N-glycoproteins
-
-
?
Oryzias latipes glycophosphoprotein MU-1 + H2O
?
show the reaction diagram
-
acid PNGase M from blastoderm stage 11, yolk-absorptive stage
-
-
?
Oryzias latipes glycophosphoprotein MU-2 + H2O
?
show the reaction diagram
-
acid PNGase M from blastoderm stage 11, yolk-absorptive stage
-
-
?
ovalbumin + H2O
?
show the reaction diagram
-
deglycosylation of the heat-denatured protein
-
-
?
ovalbumin glycopeptide + H2O
aspartic acid + NH3 + N-acetylglucosamine
show the reaction diagram
ovotransferrin glycopeptide + H2O
?
show the reaction diagram
Prunus amygdalus var. dulcis
-
Gly-Leu-Ile-His-Asn(oligosaccharide)-Arg
-
-
?
PHA glycopeptide + H2O
?
show the reaction diagram
-
-
-
-
?
pineapple stem bromelain glycopeptide + H2O
?
show the reaction diagram
porcine fibrinogen + H2O
?
show the reaction diagram
-
-
-
-
?
proton-coupled folate transporter + H2O
?
show the reaction diagram
-
-
-
-
?
receptor for advanced glycation end-products isoform H-300 + H2O
?
show the reaction diagram
-
-
-
-
?
receptor for advanced glycation end-products isoform N-16 + H2O
?
show the reaction diagram
-
-
-
-
?
ribonuclease B + H2O
?
show the reaction diagram
ricin A + H2O
?
show the reaction diagram
-
deglycosylation of ricin A, the enzyme acts in complex with protein Rad23, interaction and complex formation analysis, overview
-
-
?
RNAse + H2O
?
show the reaction diagram
removes high mannose-type N-glycan from denatured RNase B, but not from native RNase B
-
-
?
RNase B + H2O
?
show the reaction diagram
RTADELTA protein + H2O
deglycosylated RTADELTA protein + ?
show the reaction diagram
-
RTADELTA is a non-toxic mutant of ricin A-chain
-
-
?
RTADELTA-transmembrane-Leu2 protein + H2O
deglycosylated RTADELTA-transmembrane-Leu2 protein + ?
show the reaction diagram
-
-
-
-
?
sialylglycopeptide + H2O
?
show the reaction diagram
asialo-, agalactosyl-, trimannosyl- and monomanosyl-sialylglycopeptides are hydrolyzed
-
-
?
taka-amylase A + H2O
?
show the reaction diagram
Prunus amygdalus var. dulcis
-
1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1
-
-
?
transferrin + H2O
?
show the reaction diagram
Prunus amygdalus var. dulcis
-
desialylated, human
-
-
?
truncated RNaseB + H2O
deglycosylated RNaseB
show the reaction diagram
-
-
-
?
tyrosinase + H2O
?
show the reaction diagram
XylMan3FucGlcNAc2-Asn-peptide from horse raddish peroxidase + H2O
?
show the reaction diagram
-
-
-
-
?
yeast carboxypeptidase + H2O
?
show the reaction diagram
-
deglycosylation of heat-denatured protein
-
-
?
additional information
?
-
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
ATPase p97 + H2O
?
show the reaction diagram
Q9JI78
a cofactor-binding motif of p97 contained within the last 10 amino acid residues of the C terminus is both necessary and sufficient to mediate interactions of p97 with PNGase. Phosphorylation of p97s highly conserved penultimate tyrosine residue, which is the main phosphorylation site during T cell receptor stimulation, completely blocks binding of either PNGase or Ufd3 to p97. This observation suggests that phosphorylation of this residue modulates endoplasmic reticulum-associated protein degradation activity by discharging substrate-processing cofactors
-
-
?
class I MHC HC + H2O
?
show the reaction diagram
-
the enzyme is involved proteasomal degradation of glycosylated type I membrane protein class I MHC heavy chain, dislocated from ER to cytosol by cytomegalovirus-encoded glycoprotein US2 protein, overview
-
-
?
concanavalin A-precursor + H2O
?
show the reaction diagram
-
deglycosylation leads to conversion into an active lectin
-
-
?
melanopsin + H2O
?
show the reaction diagram
Q5XI55
-
-
-
?
N-glycoprotein + H2O
?
show the reaction diagram
-
PNGase is involved in the release of N-glycans from N-glycoproteins
-
-
?
Oryzias latipes glycophosphoprotein MU-1 + H2O
?
show the reaction diagram
-
acid PNGase M from blastoderm stage 11, yolk-absorptive stage
-
-
?
Oryzias latipes glycophosphoprotein MU-2 + H2O
?
show the reaction diagram
-
acid PNGase M from blastoderm stage 11, yolk-absorptive stage
-
-
?
proton-coupled folate transporter + H2O
?
show the reaction diagram
-
-
-
-
?
tyrosinase + H2O
?
show the reaction diagram
-
the enzyme is required for processing of a class I-restricted epitope from tyrosinase together with the cooperative action of endoplasmic reticulum aminopeptidase 1 and cytosolic protease, overview
-
-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
-
activity stimulating
Mn2+
-
partial activity stimulation
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
-
20% inhibition at 100 mM
benzyloxycarbonyl-VAD-fluoromethyl ketone
-
-
benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone
-
covalently bind to the active site Cys191 of recombinant yeast peptide N-glycanase
Ca2+
-
2 mM, 19% inhibition
carbobenzyloxy-Val-Ala-Asp
carbobenzyloxy-Val-Ala-Asp-alpha-fluoromethylketone
CuSO4
-
nearly complete inhibition of the acid PNGase M by 5 mM, 10-20% remaining activity at neutral PNGase M by 5 mM
Fbs1
-
Fbs1 captures malfolded glycoproteins, protecting them from the attack of PNGase, during the chaperoning or ubiquitinating operation in the cytosol
-
Fe2+
-
2 mM, 90% inhibition
FeCl3
-
nearly complete inhibition of the acid PNGase M by 5 mM, 10-20% remaining activity at neutral PNGase M by 5 mM
fucose residue alpha-1-3-linked or alpha-1-6-linked to the proximal GlnNAc residue
-
complete inhibition
-
Glycoasparagine
K+
-
significant inactivation by 100 mM
L-hyosphorin derived nonapeptide
-
1 mM causes 92% inbition of acid PNGase M
-
liberated glycan reaction product
Man8GlcNAc2-iodoacetoamide
-
-
Man9GlcNAc2-iodoacetoamide
-
strong inhibitor, irreversibly inhibits that catalytic Cys in a highly specific manner
Mg2+
-
2 mM, 14% inhibition
Monoiodoacetic acid
N,N'-diacetylchitobiose
N,N'-diacetylchitobiosylbromoacetamide
-
-
N,N'-diacetylchitobiosylchloroacetamide
-
-
N,N'-diacetylchitobiosyliodoacetamide
-
-
N-benzyloxycarbonyl-VAD-fluoromethylketone
Z-VAD-fmk
N-ethylmaleimide
Na+
-
significant inactivation by 100 mM
p-chloromercuribenzoic acid
-
0.5 mM causes 89% inhibition of neutral PNGase M
phosphate buffers
-
no significant activity
-
SDS
-
complete inactivation by 0.05% SDS can be corrected by the addition of 1% Nonidet P-40
triomannose
Tris-HCl
-
no significant activity
Triton X100
-
40% inhibition at 1%
Urea
-
80% inhibition at 2 M
yeast mannan
-
-
-
Z-VAD-fmk
-
i.e. carbobenzyloxy-Val-Ala-Asp-alpha-fluoromethylketone, a broad-spectrum caspase inhibitor, potent inhibition, binding structure determination and analysis with wild-type and mutant C191A enzyme, the inhibitor binds covalently to the catalytic residue Cys191, but not to the catalytic His218
ZnCl2
-
10-20% remaining activity at neutral PNGase M by 5 mM
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.2
asialo fetuin glycopeptide I
-
-
-
0.871
asiolo-ovomucoid glycopeptide
-
-
-
1
Beta-aspartylglycosylamine
-
pH 5.5
4
bromelain glycopeptide
Prunus amygdalus var. dulcis
-
stem bromelain undecapeptide, glycopeptidase group C
-
2 - 2.3
bromelain undecapeptide
0.114 - 1.46
Fetuin glycopeptide
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0046 - 0.05
carbobenzyloxy-Val-Ala-Asp-alpha-fluoromethylketone
0.0016
Man8GlcNAc2-iodoacetoamide
Saccharomyces cerevisiae
-
-
0.17
Man9GlcNAc2
Saccharomyces cerevisiae
-
-
0.0017
Man9GlcNAc2-iodoacetoamide
Saccharomyces cerevisiae
-
-
0.0028
N,N'-diacetylchitobiosylbromoacetamide
Saccharomyces cerevisiae
-
-
0.019
N,N'-diacetylchitobiosylchloroacetamide
Saccharomyces cerevisiae
-
-
0.0008
N,N'-diacetylchitobiosyliodoacetamide
Saccharomyces cerevisiae
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00275
-
-
0.019
Prunus amygdalus var. dulcis
-
glycopeptidase group A
0.0538
-
di-dansyl fetuin glycopeptide
0.0623
Prunus amygdalus var. dulcis
-
glycopeptidase group C
0.0855
Prunus amygdalus var. dulcis
-
glycopeptidase group B
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.2
Prunus amygdalus var. dulcis
-
-
5.3
Prunus amygdalus var. dulcis
-
taka-amylase A
6.8
-
spleen PNGase
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2 - 3.5
-
remarkable loss of activity above pH 3.5, narrow pH range, profile overview
2.5 - 7.5
Prunus amygdalus var. dulcis
-
-
3 - 8.6
-
80% of the maximum activity at pH 3.3, 65% of the maximum activity at pH 8.6
3 - 5.5
-
-
3 - 6
enzyme activity declines rapidly above pH 6.0
3 - 7.5
-
activity range, profile overview
3.5 - 4
-
acid PNGase M, whole embryo, stage 18-28
4 - 7.5
-
pH 4.0: about 50% of maximal activity, pH 7.5: about 40% of maximal activity, surface-displayed PNGase F
4 - 8.5
-
-
5 - 10
-
pH 5: about 50% of maximal activity, pH 10.0: about 70% of maximal activity
6 - 9
-
pH 6: about 70% of maximal activity, pH 9.0: about 60% of maximal activity
6.5 - 7
-
no activity below 5.5
6.8 - 7
-
-
7.5 - 8.5
-
neutral PNGase M, middle blastula blastoderm, stage 7-13
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
-
room temperature
35
-
recombinant enzyme
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15 - 25
-
remaining activity 50% at 5C and 60% at 37C
20 - 45
-
20C: about 50% of maximal activity, 45C: about 60% of maximal activity, surface-displayed PNGase F
20 - 37
-
20C: about 60% of maximal activity, 37C: about 55% of maximal activity
20 - 40
-
20C: about 80% of maximal activity, 40C: about 75% of maximal activity
20 - 60
enzyme activity declines rapidly above 40C (80% activity at 50C, 55% activity at 60C, 30% activity at 70C, no activity at 80C)
25 - 37
-
-
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
the highest activities are found in the youngest parts of the plant: apical buds, flowers and leaf blades
Manually annotated by BRENDA team
-
C3H mouse-derived L-929 fibroblast cells
Manually annotated by BRENDA team
-
the highest activities are found in the youngest parts of the plant: apical buds, flowers and leaf blades
Manually annotated by BRENDA team
-
the highest activities are found in the youngest parts of the plant: apical buds, flowers and leaf blades
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
secretory enzyme
-
Manually annotated by BRENDA team
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35500
-
gel filtration
43000
-
gel filtration
66800
Prunus amygdalus var. dulcis
-
gel filtration
69000
-
gel filtration, native enzyme
73000
Prunus amygdalus var. dulcis
-
MALDI-MS, native enzyme
79500
-
estimation by HPLC, from a standard curve of proteins of known molecular mass
80000
-
gel filtration
150000
-
estimation for acid PNGase M by gel filtration
212000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
no glycoprotein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified wild-type PUB domain, also as selenomethionine variants, by sitting drop vapour diffusion method at 17C, with a reservoir solution containing 0.2 M sodium acetate, 0.1 M Tris, pH 8.5, 30% PEG 4000, using 20% glycerol as cryoprotectant, X-ray diffraction structure determination and analysis at 1.6 A resolution, mutant L66M/L75M/L87M PUB domain are crystallized in 50% PEG 400, 0.1 M CHES, pH 9.5, 0.2 M NaCl, molecular replacement, X-ray diffraction structure determination and analysis at 1.9 A resolution
-
hanging drop vapor diffusion, crystal structure of the N-terminal domain of PNGase in complex with the cofactor-binding motif of p97 contained within the last 10 amino acid residues of the C terminus provides detailed insight into the interaction between p97 and its substrate-processing cofactors
-
purified recombinant enzyme core domain and XPCB domain in complex with the recombinant murine HR23B protein, 9.5 mg/ml of enzyme domains in a 1:1 ratio, vapour diffusion method, against reservoir solution containing 0.1 M Tris-HCl, pH 8.5, 28-32% PEG 4000, 0.2 M sodium acetate, heavy atom derivatization by soaking in 1 mM ethyl mercury thiosalicylate, 1 mM K2[PtCN4], or 1 mM KAuCN2 for 4 h, cryoprotection by 20% glycerol, X-ray diffraction structure determination and analysis at 1.85 A resolution
-
the crystal structure of PNGase in complex with N,N'-diacetylchitobiose is described, refined at 3.4 A
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
stable up to 24 h, unstable at temperatures above 30C after 1-3 h
37 - 45
-
Png1p is inactive at 37C. In contrast, the Png1p-Rad23p complex still possesses enzymatic activity at 45C
62
-
incubation for 8 h retains 65% of the original activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the recombinantly expressed enzyme is rapidly degraded after Escherichia coli cell lysis in Tris/HCl buffer which is commonly used in cell disruption procedures, although a protease inhibitor is added
-
zinc binding stabilizes the enzyme conformation by stabilizing the intermediate state and promoting product release
-
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
readily inactivated by SDS
-
288925
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
2 months at -20C, activity loss less than 50%
Prunus amygdalus var. dulcis
-
active fractions stored at -70C
-
several weeks at 4C
Prunus amygdalus var. dulcis
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
14500fold
Prunus amygdalus var. dulcis
-
460fold
-
glutathione-Sepharose column chromatography
-
native enzyme by Fe2+ affinity and phosphopeptide affinity chromatography to over 95% purity
-
Ni-nitrilotriacetate column chromatography
recombinant full-length enzyme, enzyme core domain, and enzyme XPCB domain from Escherichia coli strain BL21(DE3),the XPCB domain by chitin affinity chromatographyand gel filtration
-
recombinant GST-tagged proteasome components HR23B, S4, and AMFR from Escherichia coli by glutathione affinity batch method, recombinant His6-tagged wild-type enzyme and truncated variants from Escherichia coli by nickel affinity chromatography
-
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinit chromatography
-
recombinant His-tagged enzyme PNGase Yl from Pichia pastoris strain GS115 by ultracentrifugatio, dialysis, and nickel affinity chromatography
-
recombinant wild-type and mutant enzyme fusion PUB domain from Escherichia coli strain C41(DE3) by nickel affinity chromatography, ion exchange chromatography, and gel filtration
-
recombinant wild-type and mutant Png1 from Escherichia coli strain BL21(DE3)
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
co-expression of His-tagged or GST-tagged truncated enzyme variants and of GST-tagged enzyme mutants with His-tagged p97, transient co-expression and complex formatin of N-terminal GFP-tagged PNGase and c-myc-tagged AMFR in COS-1 cells
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co-expression of His6-tagged full-length enzyme or truncated versions with full-length Rad23 Escherichia coli strain BL21(DE3)
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co-expression of the His6-tagged wild-type enzyme and truncated variants with GST-tagged murine proteasome components HR23B, S4, and autocrine motility factor receptor AMFR in Escherichia coli
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DNA and amino acid sequence determination and analysis, recombinant expression of the His-tagged enzyme in Escherichia coli strain BL21(DE3)
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expressed in Escherichia coli BL21 cells
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expressed in Escherichia coli BL21(DE3) Codon-Plus cells
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expressed in Escherichia coli strain JM109 and in Pichia pastoris strain GS115
expressed in Saccharomyces cerevisiae
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expressed in Sf21 insect cells
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expression in Escherichia coli
expression in Escherichia coli and CHO cells
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expression in Escherichia coli as inclusion bodies
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expression of HA-tagged wild-type and mutant enzymes in U373 astrocytoma cells
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expression of His- or FLAG-tagged wild-type and mutant enzymes, co-expression with Rad23
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expression of the enzyme in U373 astrocytoma cells
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expression of wild-type and mutant enzyme PUB domain, comprising residues 11-109, fused to the lipoyl domain of Bacillus stearothermophilus dehihydrolipoamide acetyltransferase in Escherichia coli strain C41(DE3)
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gene Ddpngase, single copy gene, DNA and amino acid sequence determination and analysis, phylogenetic analysis, recombinant expression under control of the constitutive promoter actin 15 and fused to enhanced yellow fluorescent protein (EYFP), in the mutant axenic strain Ax2
gene png1, expression of wild-type and mutant Png1 in Escherichia coli strain BL21(DE3)
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importantly, heterologous expression of AtPNG1 restores N-glycanase activity in a PNGase-deficient Saccharomyces cerevisiae mutant
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into the vector pET28a for expression in Escherichia coli BL21DE3 Codon Plus RIL cells
overexpression of the full-length enzyme, enzyme core domain, and enzyme XPCB domain in Escherichia coli strain BL21(DE3)
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PNGase Yl, DNA and amino acid sequence determination and analysis, phylogenetic analysis, recombinant expression of His-tagged enzyme in Pichia pastoris strain GS115
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the PNGase F gene from Flavobacterium meningosepticum is cloned into plasmid pYD1, which enables regulated expression, secretion and detection. The expression of PNGase F gene at extracellular surface of Saccharomyces cerevisiae is confirmed by immunofluorescence microscopy. Fluorescence activated cell sorter analysis indicates that, after 36 h cultivation, 47.6% of the cell surface is anchored with target proteins. The surface engineered enzyme is confirmed to be active and reached its highest level after induced for 36 h
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C251A
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the mutant exhibits a defect in degrading RTADELTA-transmembrane-Leu2 protein. Expression of the catalytic mutant results in significant stabilization of RTADELTA protein by binding to N-glycans on the substrate
L66M/L75M/L87M
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site-directed mutagenesis, the PUB domain mutant shows slightly reduced p97 binding
C306A
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site-directed mutagenesis, inactive mutant, inhibitor Z-VAD-fmk does not bind to the mutant enzyme
G79/F80A
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site-directed mutagenesis, the double mutation completely abolishes the interaction of PNGase with p97
N41P
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site-directed mutagenesis, the mutation completely abolishes the interaction of PNGase with p97
N58A
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site-directed mutagenesis, the mutation does not affect the interaction of PNGase with p97
A208C
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inactive
A208C/Y235H
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inactive
Y235H
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inactive
C165T
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inactive
C165T/N166V/R167C
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inactive
C221T
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mutant with more than 50% wild type activity
D179E/P180A
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mutant with more than 50% wild type activity
D208R/V209A
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mutant with 10-50% wild type activity
D217A
mutation in the peptide binding site
E185R
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mutant with 10-50% wild type activity
E185R/T186V
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mutant with 10-50% wild type activity
E193D
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inactive
E193D/W194F
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inactive
E222A
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no enzymic activity
E238A
mutation in the chitobiose binding site
F224Y
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mutant with more than 50% wild type activity
G206D/L207I
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mutant with 10-50% wild type activity
H218F
mutation in the chitobiose binding site
H218Y
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site-directed mutagenesis, the mutant is inactive in protein glycosylation, but interacts with protein Rad23
I181W
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mutant with more than 50% wild type activity
I181W/K182Q
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inactive
K182Q
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mutant with more than 50% wild type activity
K253A
mutation in the chitobiose binding site
L198V
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mutant with more than 50% wild type activity
L200M
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mutant with more than 50% wild type activity
L200M/I201L
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mutant with more than 50% wild type activity
N166V/R167C
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the mutant shows 79% of wild type activity
N178A
mutation in the peptide binding site
N178T
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mutant with 10-50% wild type activity
N214C/R215Q
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mutant with 10-50% wild type activity
N266F/F227H
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mutant with more than 50% wild type activity
Q239A
mutation near the nonreducing end of the chitobiose, possible mannose binding site
Q243A
mutation near the nonreducing end of the chitobiose, possible mannose binding site
R176A
mutation in the peptide binding site
R187K/K188R
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mutant with more than 50% wild type activity
R210A
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no enzymic activity
V219L
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mutant with 1-10% wild type activity
W123A
mutation in the peptide binding site
W194F/C195A
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mutant with 10-50% wild type activity
W220F
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81% of activity compared to wild type
W231F
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83% of activity compared to wild type
W251A
mutation in the chitobiose binding site
Y211W
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mutant with more than 50% wild type activity
Y223I
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mutant with 10-50% wild type activity
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
synthesis
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PNGase F can be utilized for glycosylation of non-glycosylated recombinant proteins produced in prokaryotic cells