Information on EC 5.1.3.8 - N-Acylglucosamine 2-epimerase

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

EC NUMBER
COMMENTARY hide
5.1.3.8
-
RECOMMENDED NAME
GeneOntology No.
N-Acylglucosamine 2-epimerase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
N-Acyl-D-glucosamine = N-acyl-D-mannosamine
show the reaction diagram
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-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
epimerization
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Amino sugar and nucleotide sugar metabolism
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N-acetylneuraminate and N-acetylmannosamine degradation II
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SYSTEMATIC NAME
IUBMB Comments
N-Acyl-D-glucosamine 2-epimerase
Requires catalytic amounts of ATP.
CAS REGISTRY NUMBER
COMMENTARY hide
37318-34-6
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SWISSPROT
Manually annotated by BRENDA team
gene BACOVA_01816
UniProt
Manually annotated by BRENDA team
gene BACOVA_01816
UniProt
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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-
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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
evolution
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
N-acetyl-D-glucosamine
N-acetyl-D-mannosamine
show the reaction diagram
N-acetyl-D-mannosamine
N-acetyl-D-glucosamine
show the reaction diagram
N-Acyl-D-glucosamine
N-Acyl-D-mannosamine
show the reaction diagram
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
N-acetyl-D-glucosamine
N-acetyl-D-mannosamine
show the reaction diagram
N-acetyl-D-mannosamine
N-acetyl-D-glucosamine
show the reaction diagram
N-Acyl-D-glucosamine
N-Acyl-D-mannosamine
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5,5'-dithiobis(2-nitrobenzoic acid)
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0.1 mM, more than 95% inhibition
5,5'-dithiobis-2-nitrobenzoate
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0.1 mM, more than 95% inhibition
AMP
slight inhibition
Monoiodoacetic acid
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10 mM, more than 95% inhibition
N-acetylneuraminic acid
N-ethylmaleimide
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NEM, strong inhibitor in absence of ATP at 0.1-0.3 mM, at 5mM inhibition also in presence of ATP
NaN3
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10 mM, 18% inhibition
NEM
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1 mM, more than 95% inhibition
pyruvate
renin
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the 1:1 renin/enzyme complex has only 2% of the activity of the free enzyme
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
AMPPNP
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20 fold increase in activity
dCTP
low activating effect
dTTP
low activating effect
renin
activates isozyme BoAGE2, unrelatedly with the unique native N-terminal tag, an addiional 27-amino acid tag sequence, detagged enzyme is also activated by renin. BoAGE2 can be considered a renin-binding protein
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2 - 231.9
N-acetyl-D-glucosamine
4.76 - 57.33
N-acetyl-D-mannosamine
3.4 - 7.4
N-acetylglucosamine
1.7 - 9
N-acetylmannosamine
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.17 - 4.96
N-acetyl-D-glucosamine
300
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
14.7 - 38.7
N-acetylneuraminic acid
412 - 463
pyruvate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.07818
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AGE, immobilized recombinant enzyme, the activity yield of immobilized enzyme is approximately 30% of the free enzyme, activity is determined in 1 ml of reaction solution (100 mM Tris-HCl, pH 7.5, 100 mM GlcNAc, 10 mM MgCl2, and 2.5 mM ATP)
0.49
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specific activity in crude extract after expression in Escherichia coli cells, activity is determined in 1 ml of reaction solution (100 mM Tris-HCl, pH 7.5, 100 mM GlcNAc, 10 mM MgCl2, and 2.5 mM ATP)
5.3
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5.6
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E242Q, reverse conversion in the presence of 1 mM ATP
6.2
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R375A, reverse conversion in the presence of 1 mM ATP
6.6
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E218A, reverse conversion in the presence of 1 mM ATP
8.1
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E242Q, in the presence of 1 mM ATP
8.9
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R375K, reverse conversion in the presence of 1 mM ATP
22.8
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E218A, in the presence of 1 mM ATP
29.13
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recombinant enzyme after purification, activity is determined in 1 ml of reaction solution (100 mM Tris-HCl, pH 7.5, 100 mM GlcNAc, 10 mM MgCl2, and 2.5 mM ATP)
32
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in the presence of 1 mM ATP
35.8
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37°C, pH 7.5
46.5
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E218D, reverse conversion in the presence of 1 mM ATP
50.8
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N179A, reverse conversion in the presence of 1 mM ATP
54.8
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R375A, in the presence of 1 mM ATP
79.2
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with N-acetyl-D-glucosamine, pH 6.0, 30°C
82.1
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with N-acetyl-D-glucosamine, pH 7.0, 30°C
82.4
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with 250 mM N-acetyl-D-glucosamine, pH 9.0, 30°C
85.9
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C370A, reverse conversion in the presence of 1 mM ATP
90.8
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with N-acetyl-D-glucosamine, pH 8.0, 30°C
94.3
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R375K, in the presence of 1 mM ATP
117
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with N-acetyl-D-glucosamine, pH 7.5, 30°C
123.8
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wild type, reverse conversion in the presence of 1 mM ATP
124
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in the presence of 1 mM ATP
131
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with 430 mM N-acetyl-D-glucosamine, pH 9.0, 30°C
256.4
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E218D, in the presence of 1 mM ATP
258.5
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N179A, in the presence of 1 mM ATP
351
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C370A, in the presence of 1 mM ATP
525.8
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wild type, in the presence of 1 mM ATP
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8
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7 - 8
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7.4
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8.5
both reaction directions
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.3 - 8.3
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6.3: about 50% of maximal activity, 8.3: about 80% of maximal activity
6.5 - 9.5
activity range in both reaction directions
7 - 9.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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assay at
45
both reaction directions
47
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 60
activity range, activity increases up to 60°C
35 - 55
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more than 90% of maximal activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
42000
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calculated, SDS-PAGE
82000
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gel filtration
85000
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gel filtration
93000
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sedimentation equilibrium
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
of its apoform
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enzyme expressed in Escherichia coli
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hanging drop vapor diffusion method, structure determination by the multiple isomorphous replacement method
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 8.5
50% activity remaining within this range after 24h
727105
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
hydrolysis of wild-type and mutant enzymes by thermolysin in absence of ATP, no hydrolysis in presence of ATP
not stable to freezing and thawing
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stable to dialysis
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 20 mM potassium phosphate buffer, pH 7.6, 1.0 mM EDTA, 0.05% 2-mercaptoethanol, 5.0% sucrose, stable for at least 6 months
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stored in ice, stable for ar least 1 week
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by FP-based affinity chromatography
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of the recombinant protein by immobilized nickel-ion chromatography
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of the recombinant proteins, wild type and mutants by immobilized nickel-ion chromatography
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recombinant enzyme
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recombinant His-tagged enzyme from Escherichia coli strain Rosetta (DE3)pLys by ultrafiltration and nickel affinityy chromatography
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 recombinant Escherichia coli strains capable of expressing N-acetyl-D-glucosamine 2-epimerase and N-acetyl-D-neuraminic acid aldolase are constructed based on a highly efficient temperature-responsive expression system which is safe compared to chemical-induced systems and coupled in N-acetyl-D-neuraminic acid production
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construction of a series of chimeric enzymes successively replacing the three domains of the human enzyme - N-terminal, middle, and C-terminal - with the corresponding domains of the rat enzyme. Chimeras are expressed in Escherichia coli JM109 under the control of the Taq promoter
construction of several C-terminal deletion and multi-cysteine/serine mutants and expression in Escherichia coli
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expressed in Escherichia coli JM109 under the transcriptional control of taq promoter
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expression in Escherichia coli
expression in Jurkat cells
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expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3) in inclusion bodies, subcloning in Escherichia coli strain DH5alpha
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expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha
expression of wild-type enzyme and mutant enzymes C41S, C66S, C104S, C125S, C210S, C239S, C302S, C380S, C386S and C390S in Escherichia coli
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gene BACOVA_01816, expression of His-tagged enzyme in Escherichia coli strain Rosetta (DE3)pLys
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C370A
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site directed mutagenesis
E218A
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site directed mutagenesis
E218D
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site directed mutagenesis
E242Q
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site directed mutagenesis
E308A
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site directed mutagenesis
E308D
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site directed mutagenesis
H239A
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site directed mutagenesis
H239N
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site directed mutagenesis
H372A
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site directed mutagenesis
H372I
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site directed mutagenesis
H372N
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site directed mutagenesis
N179A
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site directed mutagenesis
R375A
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site directed mutagenesis
R375K
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site directed mutagenesis
R57A
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site directed mutagenesis
R57K
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site directed mutagenesis
C104S
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specific activity in the extract is 25% of that of the wild-type enzyme
C125S/C210S
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mutant enzyme shows 54.8% of the activity relative to the wild-type enzyme
C125S/C210S/C239S
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mutant enzyme shows 49.3% of the activity relative to the wild-type enzyme
C125S/C210S/C239S/C203S
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mutant enzyme shows 28.7% of the activity relative to the wild-type enzyme
C125S/C210S/C239S/C203S/C386S
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mutant enzyme shows 23.8% of the activity relative to the wild-type enzyme
C125S/C210S/C302S/C390S
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no activity detected
C125S/C386S
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mutant enzyme shows 68.7% of the activity relative to the wild-type enzyme
C125S/C390S
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mutant enzyme shows 5.1% of the activity relative to the wild-type enzyme
C210S
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relative specific activity in the extract is nearly the same to that of the wild-type enzyme
C210S/C386S
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mutant enzyme shows 88.7% of the activity relative to the wild-type enzyme
C210S/C390S
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mutant enzyme shows 30.9% of the activity relative to the wild-type enzyme
C239S
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relative specific activity in the extract is nearly the same to that of the wild-type enzyme
C239S/C386S
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mutant enzyme shows 116% of the activity relative to the wild-type enzyme
C239S/C390S
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mutant enzyme shows 27.8% of the activity relative to the wild-type enzyme
C302S
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relative specific activity in the extract is nearly the same to that of the wild-type enzyme
C302S/C386S
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mutant enzyme shows 65.8% of the activity relative to the wild-type enzyme
C302S/C390S
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mutant enzyme shows 7.4% of the activity relative to the wild-type enzyme
C380S
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no enzyme activity detected in the extract
C41S/C125S
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mutant enzyme shows 17.7% of the activity relative to the wild-type enzyme
C41S/C125S/C210S
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mutant enzyme shows 28.1% of the activity relative to the wild-type enzyme
C41S/C125S/C210S/C239S
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mutant enzyme shows 9.7% of the activity relative to the wild-type enzyme
C41S/C125S/C210S/C239S/C302S
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no activity detected
C41S/C125S/C210S/C239S/C302S/C386S
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no activity detected
C41S/C125S/C210S/C239S/C302S/C390S
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no activity detected
C41S/C386S
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mutant enzyme shows 0.7% of the activity relative to the wild-type enzyme
C41S/C390S
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no activity detected
C66S/C125S
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mutant enzyme shows 39.4% of the activity relative to the wild-type enzyme
C66S/C125S/C210S
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mutant enzyme shows 23.9% of the activity relative to the wild-type enzyme
C66S/C125S/C210S/C239S
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mutant enzyme shows 58.4% of the activity relative to the wild-type enzyme
C66S/C125S/C210S/C239S/C302S
-
mutant enzyme shows 15.5% of the activity relative to the wild-type enzyme
C66S/C125S/C210S/C302S/C386S
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no activity detected
C66S/C125S/C210S/C302S/C390S
-
no activity detected
C66S/C386S
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mutant enzyme shows 113% of the activity relative to the wild-type enzyme
C66S/C390S
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mutant enzyme shows 14.4% of the activity relative to the wild-type enzyme
DELTA380-417
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mutant enzyme has no activity
DELTA386-417
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mutant enzyme has no activity
DELTA390-417
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mutant enzyme has no activity
DELTA400-417
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C-terminal deletion mutant has approximately 50% activity relative to the wild-type enzyme
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant His6-tagged enzyme expressed in Escherichia coli strain BL21(DE3) cannot be solubilized/purified from inclusion bodies
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
synthesis
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