Information on EC 2.7.7.43 - N-acylneuraminate cytidylyltransferase

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

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EC NUMBER
COMMENTARY hide
2.7.7.43
-
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RECOMMENDED NAME
GeneOntology No.
N-acylneuraminate cytidylyltransferase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
CTP + N-acetylneuraminate = diphosphate + CMP-N-acylneuraminate
show the reaction diagram
CTP + N-acylneuraminate = diphosphate + CMP-N-acylneuraminate
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
nucleotidyl group transfer
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
CMP-N-acetylneuraminate biosynthesis I (eukaryotes)
-
-
CMP-N-acetylneuraminate biosynthesis II (bacteria)
-
-
metabolism of amino sugars and derivatives
-
-
Amino sugar and nucleotide sugar metabolism
-
-
Metabolic pathways
-
-
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SYSTEMATIC NAME
IUBMB Comments
CTP:N-acylneuraminate cytidylyltransferase
Acts on N-acetyl- and N-glycolyl- derivatives.
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CAS REGISTRY NUMBER
COMMENTARY hide
9067-82-7
-
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
wild-type and glycosylation mutant LEC29.Lec32, the mutation resides in either the structural gene encoding CMP-NeuAc synthetase or in a gene that regulates the prpduction of the active enzyme
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
O18:K1
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
group B, high-producing type Ib strain
-
-
Manually annotated by BRENDA team
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
-
CMP-sialic acid synthetase is a key enzyme in the biosynthesis of the capsular polysaccharides required for bacterial infection
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
4-O-methyl-N-acetylneuraminate + CTP
CMP-4-O-methyl-N-acetylneuraminate + diphosphate
show the reaction diagram
CDP + N-acetylneuraminate
phosphate + CMP-N-acetylneuraminate
show the reaction diagram
CTP + (2S,4S,5R,6R)-6-((1R,2S)-3-azido-1,2-dihydroxy-propyl)-2,4,5-trihydroxy-6-methyl-tetrahydro-pyran-2-carboxylic acid
diphosphate + CMP-(2S,4S,5R,6R)-6-((1R,2S)-3-azido-1,2-dihydroxy-propyl)-2,4,5-trihydroxy-6-methyl-tetrahydro-pyran-2-carboxylic acid
show the reaction diagram
-
-
-
-
?
CTP + (N-4-O-)diacetylneuraminic acid
diphosphate + CMP-N-4-O-diacetylneuraminate
show the reaction diagram
-
(N-4-O-)diacetylneuraminic acid is 46% effective compared to N-acylneuraminate
-
-
?
CTP + 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid
diphosphate + CMP-2-keto-3-deoxy-D-glycero-D-galacto-nononic acid
show the reaction diagram
CTP + 3-deoxy-D-galacto-2-octulosonic acid
diphosphate + CMP-3-deoxy-D-galacto-2-octulosonic acid
show the reaction diagram
-
-
-
-
?
CTP + 3-deoxy-D-glycero-D-galacto-nononate
diphosphate + CMP-3-deoxy-D-glycero-D-galacto-nononate
show the reaction diagram
CTP + 5-deaminoneuraminate
diphosphate + CMP-5-deaminoneuraminate
show the reaction diagram
-
-
-
?
CTP + 8-O-methyl-N-acetylneuraminate
diphosphate + CMP-(8-O-methyl)-N-acetylneuraminate
show the reaction diagram
-
-
-
-
?
CTP + deaminoneuraminic acid
?
show the reaction diagram
-
-
-
-
?
CTP + N-(N-benzyloxycarbonyl-glycyl)-muramic acid
diphosphate + CMP-N-(N-benzyloxycarbonyl-glycyl)-muramic acid
show the reaction diagram
-
-
-
-
?
CTP + N-acetyl-neuraminate
diphosphate + CMP-N-acetylneuraminate
show the reaction diagram
-
-
-
?
CTP + N-acetyl-neuraminic acid
CMP-Neu5Ac + diphosphate
show the reaction diagram
-
-
-
-
?
CTP + N-acetylmuramic acid
diphosphate + CMP-N-acetylmuramic acid
show the reaction diagram
-
-
-
-
?
CTP + N-acetylneuraminate
diphosphate + CMP-N-acetylneuraminate
show the reaction diagram
CTP + N-acetylneuraminic acid
diphosphate + CMP-N-acetylneuraminic acid
show the reaction diagram
CTP + N-acylneuraminate
diphosphate + CMP-N-acylneuraminate
show the reaction diagram
CTP + N-acylneuraminate methyl ester
diphosphate + CMP-N-acylneuraminate methyl ester
show the reaction diagram
CTP + N-azidoacetylmuramic acid
diphosphate + CMP-N-azidoacetylmuramic acid
show the reaction diagram
-
-
-
-
?
CTP + N-azidomuramic acid
diphosphate + CMP-N-azidomuramic acid
show the reaction diagram
-
-
-
-
?
CTP + N-glycolylneuraminate
diphosphate + CMP-N-glycolylneuraminate
show the reaction diagram
CTP + N-glycolylneuraminic acid
CMP-N-glycolylneuraminate + diphosphate
show the reaction diagram
CTP + N-hydroxyacetylmuramic acid
diphosphate + CMP-N-hydroxyacetylmuramic acid
show the reaction diagram
-
-
-
-
?
CTP + N-hydroxymuramic acid
diphosphate + CMP-N-hydroxymuramic acid
show the reaction diagram
-
-
-
-
?
CTP + N-methylglycolylneuraminate
diphosphate + CMP-N-methylglycolylneuraminate
show the reaction diagram
-
-
-
-
?
CTP + sialic acid
CMP-sialic acid + diphosphate
show the reaction diagram
deaminoneuraminic acid + CTP
CMP-KDN + diphosphate
show the reaction diagram
N-glycolylneuraminic acid (Neu5Gc) and deaminoneuraminic acid (KDN) are also substrates
-
-
?
fluoroacetylneuraminate + CTP
diphosphate + CMP-N-fluoroacetylneuraminate
show the reaction diagram
N-acetyl-4-O-acetylneuraminate + CTP
diphosphate + CMP-N-acetyl-4-O-acetylneuraminate
show the reaction diagram
N-acetyl-7(8)-O-acetylneuraminate + CTP
diphosphate + CMP-N-acetyl-7(8)-O-acetylneuraminate
show the reaction diagram
N-acetylneuraminic acid + CTP
CMP-Neu5Ac + diphosphate
show the reaction diagram
N-acetylneuraminic acid + CTP
CMP-NeuAc + diphosphate
show the reaction diagram
N-chloroacetylneuraminate + CTP
diphosphate + CMP-N-chloroacetylneuraminate
show the reaction diagram
N-glycolylneuraminic acid + CTP
CMP-N-glycolylneuraminate + diphosphate
show the reaction diagram
N-glycolylneuraminic acid + CTP
CMP-Neu5Gc + diphosphate
show the reaction diagram
N-glycolylneuraminic acid (Neu5Gc) and deaminoneuraminic acid (KDN) are also substrates
-
-
?
TTP + N-acylneuraminate
diphosphate + TMP-N-acylneuraminate
show the reaction diagram
TTP + N-glycolylneuraminic acid
TMP-N-glycolylneuraminate + diphosphate
show the reaction diagram
UDP + N-acetylneuraminate
phosphate + UMP-N-acetylneuraminate
show the reaction diagram
-
-
-
-
?
UDP + N-acylneuraminate
phosphate + UMP-N-acetylneuraminate
show the reaction diagram
UTP + N-acylneuraminate
diphosphate + UMP-N-acylneuraminate
show the reaction diagram
UTP + N-glycolylneuraminic acid
UMP-N-glycolylneuraminate + diphosphate
show the reaction diagram
-
-
-
-
?
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
CTP + N-acylneuraminate
diphosphate + CMP-N-acylneuraminate
show the reaction diagram
CTP + sialic acid
CMP-sialic acid + diphosphate
show the reaction diagram
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Zn2+
-
62% as effective as Mn2+; can replace Mn2+, 62% as effective as Mn2+ at 10 mM
additional information
-
Ca2+ shows no effect on enzyme activity
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(NH4)2SO4
-
1.0 M, 83% inhibition, reversible by desalting
2'-deoxy-CTP
-
0.3 mM; 44% inhibition
2-deoxy-2,3-dehydro-N-acetylneuraminate
-
4.6 mM, 33% inhibition
4-(hydroxymercuri) benzoic acid
-
-
4-(hydroxymercuri)benzoic acid
-
-
5,5'-dithiobis(2-nitrobenzoic acid)
-
-
5-Mercuri-CTP
-
-
5-mercury-CTP
-
-
ATP
-
10 mM, 60% inhibition
Ba2+
-
20 mM, less than 30% inhibition
Cd2+
-
inhibits when incubated in presence of Mg2+ at the same concentration
CMP-N-acylneuraminate
cytidine
-
60 mM, 37% inhibition
diphosphate
DTT
-
in the presence of 0.01 M DTT enzyme shows 20% activity loss
GMP
-
20 mM, 64% inhibition
iodoacetate
-
-
N-ethylmaleimide
-
-
NaF
-
in the presence of 0.06 M NaF enzyme shows 17% activity loss
NaN3
-
5 mM, 70% inhibition
Ni2+
-
20 mM in presence of 10 mM Mg2+, more than 90% inhibition
p-hydroxymercuribenzoic acid
-
-
periodate-oxidized CTP
-
-
-
PHMB
-
1 mM, complete inhibition
saturated sulfo-CDP
-
1 mM, 37°C, pH 8.5, 15 min, 55% inhibition
saturated sulfo-UDP
-
1 mM, 37°C, pH 8.5, 15 min, 35% inhibition
saturated sulfo-UDP ester
-
1 mM, 37°C, pH 8.5, 15 min, 24% inhibition
Sr2+
-
20 mM, less than 30% inhibition
sulfo-CDP
-
1 mM, 37°C, pH 8.5, 15 min, 47% inhibition
sulfo-CTP
-
1 mM, 37°C, pH 8.5, 15 min, 39% inhibition
sulfo-UDP
-
1 mM, 37°C, pH 8.5, 15 min, 6% inhibition
sulfo-UTP
-
1 mM, 37°C, pH 8.5, 15 min, 33% inhibition
TTP
-
10 mM in presence of 3 mM CTP, 71% inhibition
UDP-N-acetylglucosamine
-
10 mM, 26% inhibition
UTP
-
10 mM in presence of 3 mM CTP, 75% inhibition
additional information
-
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
ATP
-
1 mM, 20% activation
dithiothreitol
DTT
-
maximum activity in the presence of 0.2 mM DTT
additional information
-
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.56 - 3
2-keto-3-deoxy-D-glycero-D-galacto-nononic acid
0.56 - 3
3-deoxy-D-glycero-D-galacto-nononate
2.5
4-O-methyl-N-acetylneuraminate
-
pH 7, 37°C
1.5 - 5.3
8-O-methyl-N-acetylneuraminate
2.1
CDP
-
pH 9.0, 37°C, reaction with N-acetylneuraminate
0.00046 - 4
CTP
0.62
deaminoneuraminic acid
-
1.2 - 1.4
N-acetyl-4-O-acetylneuraminate
1.1 - 1.6
N-acetyl-7(8)-O-acetylneuraminate
0.00036 - 7.6
N-acetylneuraminate
1.3
N-acetylneuraminate methyl ester
-
pH 9.0, 37°C, reaction with CTP
0.26 - 2.8
N-acetylneuraminic acid
0.13 - 4
N-acylneuraminate
1.2 - 6.2
N-glycolylneuraminate
0.16 - 0.35
N-glycolylneuraminic acid
2 - 7
N-methylglycolylneuraminate
0.045 - 0.07
sialic acid
8.1
TTP
-
pH 9.0, 37°C, reaction with N-acetylneuraminate
3
UDP
-
pH 9.0, 37°C, reaction with N-acetylneuraminate
2.7
UTP
-
pH 9.0, 37°C, reaction with N-acetylneuraminate
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.9 - 21
8-O-methyl-N-acetylneuraminate
0.001 - 25
CTP
0.067
deaminoneuraminic acid
-
4.2 - 32
N-acetylneuraminate
2.25
N-acylneuraminate
-
at 50°C, 10 mM MnCl2, 50 mM Tris buffer (pH 9.5)
3.2 - 36
N-glycolylneuraminate
0.68 - 1.66
N-glycolylneuraminic acid
2.9 - 12
N-methylglycolylneuraminate
2.29 - 3.56
sialic acid
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.55 - 1.3
8-O-methyl-N-acetylneuraminate
0.0013 - 40
CTP
0.0005 - 86
N-acetylneuraminate
3.8 - 5.9
N-glycolylneuraminate
1.6 - 1.7
N-methylglycolylneuraminate
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.5
CMP
-
pH 9.0, 37°C
additional information
additional information
-
-
-
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
CDP
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0017
-
mutant enzyme DELTA1-227
0.0113
-
wild-type enzyme
0.045
-
-
0.165
-
30°C, pH 9.0
0.2
substrate N-glycolylneuraminate, pH 7.5, temperature not specified in the publication
0.3
substrate 5-deaminoneuraminate, pH 7.5, temperature not specified in the publication
0.83
-
at pH 7.6
1.58 - 3.17
-
at pH 9.0
2.7
substrate N-glycolylneuraminate, pH 7.5, temperature not specified in the publication
3.3
substrate 5-deaminoneuraminate, pH 7.5, temperature not specified in the publication
4.5
substrate N-acetylneuraminate, pH 7.5, temperature not specified in the publication
18.25
-
-
additional information
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
-
reaction with Mn2+
7.5
-
assay at
8.3 - 9.4
-
-
8.5 - 9
-
activity in presence of Mg2+
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 12.5
-
activity decreases dramatically below pH 7.0 and above pH 12.5
7 - 10
-
pH 7.0: about 90% of maximal activity, pH 10.0: about 70% of maximal activity
7.5 - 9.5
-
pH 7.5: about 20% of maximal activity, pH 8.0: about 90% of maximal activity, pH 9.5: about 65% of maximal activity
7.5 - 9
7.5 - 10.5
enzyme is active in a wide pH-range
8 - 13
-
enzyme is active between pH 8-13; in Tris-HCl buffer
8.5 - 9.5
-
-
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
-
activity assay
40
-
greatest activity is observed at 40°C
40 - 60
-
immobilized enzyme yields maximal activity at 40°C which is maintained up to 60°C, whereas the activity of the soluble enzyme decreases sharply above 40°C
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 42
-
4°C: activity is undetectable, 25-37°C: maximal activity, 42°C: 12% of maximal activity
30 - 50
-
30°C: about 40% of maximal activity, 50°C: about 90% of maximal activity
33 - 42
-
50% of maximal activity at 33°C and at 42°C
37 - 60
-
enzyme is active between 37°C - 60°C; in the presence of 0.2 mM dithiothreitol
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.6 - 5.9
-
isoelectric focusing
5.9
-
isoelectric focusing
6.5
-
chromatofocusing
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
robust expression in the entire central nervous system, the somites, the notochord, and the developing pronephric duct
Manually annotated by BRENDA team
more or less ubiquitously expressed from the end of gastrula through segmentation
Manually annotated by BRENDA team
-
DmCSAS gene is greatly enriched in adult head, suggesting a possible role in the central nervous system
Manually annotated by BRENDA team
robust expression in the entire central nervous system, the somites, the notochord, and the developing pronephric duct
Manually annotated by BRENDA team
robust expression in the entire central nervous system, the somites, the notochord, and the developing pronephric duct
Manually annotated by BRENDA team
additional information
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
unlike all vertebrate CSAS that localize to the nucleus, the DmCSAS is localized to the Golgi compartment
Manually annotated by BRENDA team
additional information
-
replacement of the N-terminal leader sequence of DmCSAS with the human CSAS N-terminal sequence results in the redirection of the chimeric CSAS protein to the nucleus but with concomitant loss of activity
-
Manually annotated by BRENDA team
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
18300
-
C-terminal domain of CMP-sialic acid synthetase, calculated
24800
-
gel filtration
24892
-
x * 24892, calculation from nucleotide sequence
24893
-
x * 24893, mass spectrometry
25400
-
gel filtration
25440
-
2 * 25440, dimer or larger oligomer, electrospray mass spectrometry
25600
-
2 * 25600, dimer or larger oligomer, SDS-PAGE
26400
-
N-terminal domain of CMP-sialic acid synthetase, calculated
27313
-
x * 27313, electrospray mass spectrometry
27500
-
predicted from cDNA
33700
-
x * 33700, SDS-PAGE
44400
-
CMP-sialic acid synthetase, calculated
48058
-
x * 48058, calculation from nucleotide sequence
48200
or pentamer, 4 * 48200, calculated; or tetramer, 5 * 48200, calculated
49000
-
SDS-PAGE
49600
4 * 49600, calculated
50000
-
x * 50000, SDS-PAGE
52000
-
3 * 52000, SDS-PAGE
55800
-
gel filtration
56600
-
dimer, N-terminal domain of CMP-sialic acid synthetase, determined by gel filtration
58000
-
2 * 58000, SDS-PAGE
60000
-
1 * 60000, SDS-PAGE
66000
-
SDS-PAGE, recombinant thioredoxin fusion protein
69000
-
SDS-PAGE, recombinant thioredoxin fusion protein; x * 69000, SDS-PAGE
87000
-
tetramer, C-terminal domain of CMP-sialic acid synthetase, determined by gel filtration
116000
-
gel filtration
135000
-
gel filtration
158000
-
gel filtration
160000
-
gel filtration
162000
-
gel filtration
164000
-
1 * 164000, SDS-PAGE
175800
-
tetramer, CMP-sialic acid synthetase, determined by gel filtration
193400
gel filtration
236100
gel filtration
additional information
-
47000-74000 Da, gel filtration
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
pentamer
or tetramer, 5 * 48200, calculated
tetramer
trimer
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POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
no carbohydrates can be detected
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
the structure of the C-terminal domain of CMP-sialic acid synthetase is solved to 1.9 A resolution
-
modeling of the closed conformation. Enzyme forms a dimer complex. Amino acids involved in the Neu5Ac binding pocket are Glu162 and Arg165 from monomer B and Gln104, Ser82, Val86, Tyr179, and Phe192 from monomer A
-
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 12.5
-
activity is drastically reduced below pH 7.0 and above pH 12.5
690583
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
-
not stable at room temperature for 12 h
35
-
60 min, completely stable
37
-
60 min, about 10% loss of activity
37 - 70
-
complete loss of activity above 70°C, no loss of activity after 24 h incubation at 37°C and 50°C
40 - 45
-
the enzyme loses complete activity after 50 and 10 min when stored at 40°C and 45°C, respectively
50
-
10 min, 85% loss of activity
55
-
20 min, complete loss of activity
70
-
the enzyme retains activity at 70°C
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme is stable in lyophilized form
-
not stable to freezing and thawing, addition of substrates partially stabilizes the purified enzyme to freezing and thawing
-
not stable to lyophilization
-
purified enzyme is sensitive to repeated freezing
-
stability can be improved by 1 mM 2-mercaptoethanol
-
stabilized against thermal inactivation by some salts, NaCl, Na2SO4 or Na2HPO4, or by glycerol
-
stable in the frozen state, each cycle of freezing and thawing leads to a 15% decrease in activity
-
the enzyme is equally stable with or without the presence of glycerol at different temperatures
-
the enzyme tolerates flash freezing and lyophilization
-
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C or -80°C, in 50% (v/v) glycerol, several months, remains stable
-
-20°C or -80°C, the relatively crude preparation is stable to storage as a 50% glycerol solution for at least several months
-
-20°C, 2 weeks, more than 50% loss of activity
-
-20°C, partially purified enzyme can be stored in 43% glycerol or in 50 mM Tris/HCl or potassium phosphate, pH 7.5, for several months without loss of activity
-
-20°C, stable for at least 1 year
-80°C, completely stable for 8 months
-
4°C, 7 months, a magnetic nanoparticle-CSS preparation retains almost 100% activity
-
4°C, above pH 7.0, stable for several weeks
-
4°C, at pH 7.0, several weeks, remains stable
-
4°C, pH 7.6, 83% of the original activity is recovered after 4 weeks
-
4°C, stable for 3 weeks
-
4°C, the relatively crude preparation is stable to storage as a 65% ammonium sulfate suspension or 20% glycerol solution for at least several months
-
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by column chromatography on Sephacryl S-200 (gel filtration), Yellow-86-Agarose (affinity) and Phenyl-Sepharose (hydrophobic affinity)
-
C-His6-tagged CSS is purified using a nickel-nitrilotriacetic acid-agarose (Ni2+-NTA-agarose) affinity column
Ni-NTA column chromatography
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partial
recombinant proteins are expressed in Escherichia coli BL21DE3 cells and purified by affinity chromatography utilizing the StrepII-Tag, further purified on a Superdex 200 HR 10/30 columnn
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wild-type enzyme and mutant enzymes
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a series of deletions from the 3'-end of the CMP-NeuAc synthetase coding region is constructed and expressed in Escherichia coli
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cloned in Escherichia coli as a His6-tagged fusion protein
cloned in Escherichia coli XL-10; expressed in Escherichia coli strain BL21
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cloning is achieved by complementation of the Chineses hamster ovary lec32 mutation that causes a deficiency in CMP-N-acetylneuraminate synthetase activity, it also causes polysialic acid to be expressed in the capsule of the CMP-Neu5Ac synthetase negative Escherichia coli mutant EV5; expression in CHO cells or Escherichia coli
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DmCSAS is cloned into the baculovirus vector pBlueBac4.5 (pBlueBac-DmCSAS4) which is used to transfect LEC29.Lec32 cells
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expressed in CHO cells, HeLa cells, and NIH-3T3 cells; expression in CHO cells
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expressed in Escherichia coli and CHO cells
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expression in Escherichia coli
expression in Escherichia coli, enzyme can function in K1 capsular polysaccharide biosynthesis in Escherichia coli
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expression in Escherichia coli; expression in Escherichia coli
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high-level expression in Escherichia coli
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human CMP-N-acetylneuraminic acid synthetase is expressed in tobacco BY2 suspension-cultured cells, enzyme is demonstrated to be active
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the enzyme is subcloned for overexpression in Escherichia coli K12 using the expression vector pKK233-3
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the gene is cloned into a T7 expression vector, the protein is expressed in Escherichia coli
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the N-terminal domain, residues 39-267, and the C-terminal domain, residues 267-432, of CMP-sialic acid synthetase, and CMP-sialic acid synthetase, residues 39-432, are cloned into a pET22b-Strep vector
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transfection of NIH-3T3 and EPC cells; transfection of NIH-3T3 and EPC cells
transformation of the CMP-NeuNAc defective Escherichia coli K1 strain EV5 with CMP-NeuNAc synthetase from Neisseria meningitidis can complement the defect in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
DELTA1-227
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mutant enzyme shows no CMP-N-acetylneuraminic acid synthetase activity, mutant enzyme is able to hydrolyze platelet activating factor
DELTA1-229
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mutant enzyme shows no CMP-N-acetylneuraminic acid synthetase activity, mutant enzyme is not able to hydrolyze platelet activating factor
DELTA230-418
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mutant enzyme shows 43% of the wild-type activity with CTP and N-acylneuraminate as substrates, decrease in stability compared to wild-type enzyme
DELTA247-418
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mutant enzyme shows 15% of the wild-type activity with CTP and N-acylneuraminate as substrates, decrease in stability compared to wild-type enzyme
DELTA340-418
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mutant enzyme shows 65% of the wild-type activity with CTP and N-acylneuraminate as substrates, as stable as wild-type enzyme
DELTA384-418
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mutant enzyme shows 31% of the wild-type activity with CTP and N-acylneuraminate as substrates, as stable as wild-type enzyme
DELTA396-418
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mutant enzyme shows 38% of the wild-type activity with CTP and N-acylneuraminate as substrates, as stable as wild-type enzyme
DELTA1-227
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mutant enzyme shows no CMP-N-acetylneuraminic acid synthetase activity, mutant enzyme is able to hydrolyze platelet activating factor
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DELTA340-418
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mutant enzyme shows 65% of the wild-type activity with CTP and N-acylneuraminate as substrates, as stable as wild-type enzyme
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DELTA384-418
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mutant enzyme shows 31% of the wild-type activity with CTP and N-acylneuraminate as substrates, as stable as wild-type enzyme
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DELTA396-418
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mutant enzyme shows 38% of the wild-type activity with CTP and N-acylneuraminate as substrates, as stable as wild-type enzyme
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DELTA408-424
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deletion mutant devoid of the second putative nuclear export signal, by immunofluorescent microscopy it is shown that this deletion mutant has an increased nuclear localisation combined with a decreased cytoplasmic localisation
DELTA61-69
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deletion mutant devoid of the first putative nuclear export signal, by immunofluorescent microscopy it is shown that is deletion mutant has an increased nuclear localisation combined with a decreased cytoplasmic localisation
K198A
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mutant enzyme is active at moderately reduced level
R199A
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inactive mutant protein
R199A/R202A
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inactive mutant protein
R201A
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mutant enzyme is active at moderately reduced level
R202A
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mutant enzyme shows drastically reduced activity
D209A
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almost complete loss of activity. Residue involved in metal binding
D211A
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dramatic loss of activity. Residue involved in metal binding
F192A
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8fold increase on Km value for CTP
F193A
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3fold increase on Km value for CTP
K142A
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10000fold reduction in kcat with an insignificant, fourfold, rise in Km for CTP; dramatic loss of activity. Residue involved in metal binding
N175A
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16fold increases in the Km value for CTP and 23fold for N-acetylneuraminate
Q104A
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40fold inccrease in Km value for N-acetylneuraminate
Q104E
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dramatic loss of activity. Residue involved in metal binding
Q104L
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dramatic loss of activity. Residue involved in metal binding
Q104N
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dramatic loss of activity. Residue involved in metal binding
Q163A
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mutant displays improved substrate promiscuity
R173A
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38fold increase in Km value for N-acetylneuraminate
S31R
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mutant displays improved substrate promiscuity, catalytic activities for substrates N-glycolylneuraminate, N-methylglycolylneuraminate and 8-O-methyl N-acetyl-neuraminate are improved compared to wild-type
Y179A
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200fold decrease in kcat value
additional information
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
agriculture
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this approach enables plant cells to produce glycoproteins with mammalian-type N-glycans
medicine
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the enzyme is involved in the production of activated sialic acids. Sialic acids are virulence factors in a variety of bacterial species, e.g. Neisseria menigitidis. As such, the identification of the bacterial CMP-NeuAc synthetase active site can serve as a starting point for rational drug design strategies
synthesis
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Show AA Sequence (797 entries)
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