Information on EC 2.5.1.56 - N-acetylneuraminate synthase

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

EC NUMBER
COMMENTARY hide
2.5.1.56
-
RECOMMENDED NAME
GeneOntology No.
N-acetylneuraminate synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
phosphoenolpyruvate + N-acetyl-D-mannosamine + H2O = phosphate + N-acetylneuraminate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
condensation
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Amino sugar and nucleotide sugar metabolism
-
-
CMP-N-acetylneuraminate biosynthesis II (bacteria)
-
-
Metabolic pathways
-
-
metabolism of amino sugars and derivatives
-
-
SYSTEMATIC NAME
IUBMB Comments
phosphoenolpyruvate:N-acetyl-D-mannosamine C-(1-carboxyvinyl)transferase (phosphate-hydrolysing, 2-carboxy-2-oxoethyl-forming)
-
CAS REGISTRY NUMBER
COMMENTARY hide
37290-66-7
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene NANS
UniProt
Manually annotated by BRENDA team
strain K1
-
-
Manually annotated by BRENDA team
strain K1-M12
-
-
Manually annotated by BRENDA team
gene NANS
SwissProt
Manually annotated by BRENDA team
gene siaC
UniProt
Manually annotated by BRENDA team
gene siaC
UniProt
Manually annotated by BRENDA team
gene MvNeuB
UniProt
Manually annotated by BRENDA team
strain 60E
-
-
Manually annotated by BRENDA team
serotype B, gene synC
UniProt
Manually annotated by BRENDA team
strain PRM102
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
formation of sialic acid or N-acetylneuraminic acid to produce a capsular polysacccharide of polysialic acid to evade the host's immune system by mimicking its own cell surface polysaccharides
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
2-acetamido-6-azido-2,6-dideoxy-D-mannose + phosphoenolpyruvate + H2O
5-acetamido-9-azido-3,5,9-trideoxy-D-glycero-D-galacto-2-nonulosonic acid + phosphate
show the reaction diagram
N-acetyl-D-galactosamine + phosphoenolpyruvate + H2O
?
show the reaction diagram
-
15% of the activity with N-acetyl-D-mannosamine
-
-
?
N-acetyl-D-mannosamine + phosphoenolpyruvate + H2O
?
show the reaction diagram
N-acetyl-D-mannosamine + phosphoenolpyruvate + H2O
N-acetylneuraminate + phosphate
show the reaction diagram
phosphoenolpyruvate + N-acetyl-D-mannosamine + H2O
N-acetylneuraminate + phosphate
show the reaction diagram
-
-
-
?
phosphoenolpyruvate + N-acetyl-D-mannosamine + H2O
phosphate + N-acetylneuraminate
show the reaction diagram
phosphoenolpyruvate + N-butanoyl-D-mannosamine + H2O
phosphate + N-butanoylneuraminate
show the reaction diagram
-
-
-
-
?
phosphoenolpyruvate + N-pentanoyl-D-mannosamine + H2O
phosphate + N-pentanoylneuraminate
show the reaction diagram
-
-
-
-
?
phosphoenolpyruvate + N-propanoyl-D-mannosamine + H2O
phosphate + N-propanoylneuraminate
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-mannosamine + phosphoenolpyruvate + H2O
?
show the reaction diagram
phosphoenolpyruvate + N-acetyl-D-mannosamine + H2O
N-acetylneuraminate + phosphate
show the reaction diagram
E0NCD4
-
-
-
?
phosphoenolpyruvate + N-acetyl-D-mannosamine + H2O
phosphate + N-acetylneuraminate
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
no metal ion requirement
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5-acetamido-4,6,7,8,9-pentahydroxy-2-phosphorylnonanoic acid ditriethylammonium salt
4.5/1 mixture of 2S-inhibitor/2R-inhibitor, competitive inhibitor against phosphoenolpyruvate
AgNO3
-
1 mM, 61% inhibition
Co2+
stabilizes at low and inhibits at higher concentrations
Fe2+
-
1 mM FeCl2, 40% inhibition
HgCl2
-
at 0.01 mM 85% inhibition, at 0.1 mM complete inhibition
hydroxylamine
-
1 mM, 23% inhibition
Iodine
-
1 mM, 30% inhibition
Li+
-
1 mM LiCl, 30% inhibition
N-bromosuccinimide
-
1 mM, 24% inhibition
PCMB
-
1 mM, complete inhibition
Phenylglyoxal
-
N-acetylmannosamine or phosphoenolpyruvate protect
Semicarbazide
-
1 mM, 20% inhibition
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
glutathione
-
required for maximal activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5.6 - 18
N-acetyl-D-mannosamine
15
N-butanoyl-D-mannosamine
-
pH 8.0, 37C
31.1
N-pentanoyl-D-mannosamine
-
pH 8.0, 37C
5.5
N-propanoyl-D-mannosamine
-
pH 8.0, 37C
0.04 - 7.3
phosphoenolpyruvate
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.83 - 19.9
N-acetyl-D-mannosamine
3.5
N-butanoyl-D-mannosamine
Campylobacter jejuni
-
pH 8.0, 37C
0.8
N-pentanoyl-D-mannosamine
Campylobacter jejuni
-
pH 8.0, 37C
11.8
N-propanoyl-D-mannosamine
Campylobacter jejuni
-
pH 8.0, 37C
3.75 - 19
phosphoenolpyruvate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0031
5-acetamido-4,6,7,8,9-pentahydroxy-2-phosphorylnonanoic acid ditriethylammonium salt
4.5/1 mixture of 2S-inhibitor/2R-inhibitor, a 1.2/1 mixture of 2S-inhibitor/2R-inhibitor requires a 2.5-fold lower total inhibitor concentration, 37C, 100 mM Tris-HCl buffer, pH 7.0, 1 mM MnCl2, 50 microM to 1 mM phosphoenolpyruvate, His-tagged enzyme, purine nucleoside phosphorylase, 200 mircoM 2-amino-6-mercapto-7-methylpurine ribonucleoside
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.000416
-
-
1.018
-
pH 8.0, 37C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.3
recombinant enzyme
8.3
-
in Tris buffer
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 12
-
pH 4.0: about 50% of maximal activity, pH 12.0: about 40% of maximal activity
6.5 - 10
-
pH 6.5: about 40% of maximal activity, pH 10.0: about 60% of maximal activity
7 - 10
activity range, inactive at pH 6.5, profile overview
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
recombinant enzyme
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 50
the relative enzyme activity is approximately 20% at 5C, and increasing to a maximum at around 30C, and decreases rapidly above 30C, no activity remains at 55C. The enzyme from a psychrophilic bacterium, MvNeuB, is less stable at higher temperature and has a high catalytic activity at lower temperature compared to mesophilic counterparts. MvNeuB is a typical cold adapted enzyme
20 - 60
-
about 60% of maximal activity at 20C and at 60C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38629
-
1 * 38629, calculated from amino acid sequence
38630
-
calculated from amino acid sequence
38649
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1 * 38649, ESI-MS
38987
-
2 * 38987, electrospray ionisation mass spectrometry
39000
-
2 * 39000, SDS-PAGE
40538
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4 * 40538, LC-MS analysis
43300
-
x * 43300, His-tagged recombinant enzyme, MALDI-TOF MS
52000
-
2 * 52000, SDS-PAGE
74000
recombinant enzyme, gel filtration
95000
gel filtration
106000
-
gel filtration
135000
-
recombinant protein containing a hexahistidine tag at the N-terminus, gel filtration
160000
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 43300, His-tagged recombinant enzyme, MALDI-TOF MS
homodimer
monomer
-
1 * 38629, calculated from amino acid sequence; 1 * 38649, ESI-MS
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by small angle X-ray scattering and structure analysis, PDB ID 1XUZ
-
malate-bound enzyme and with phosphoenolpyruvate or the substrate analogue N-acetylmannosaminitol
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purified and concentrated enzyme crystallized at 18C, hanging-drop vapor diffusion, 10 mM MnCl2, 1.50-1.55 M malic acid, pH 6.2, soaked in 2 M sodium phosphate, pH 6.2, with 10 mM MnCl2, 25% ethylene glycol, frozen in liquid nitrogen at -173C, complex with Mn2+ and 4.5/1 mixture of 2S-inhibitor/2R-inhibitor
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
-
25C, 1 h, about 50% loss of activity
637321
5
-
25C, 1 h, about 40% loss of activity
637321
6
-
25C, 1 h, about 25% loss of activity
637321
7 - 10
-
25C, 1 h, stable
637321
8
purified recombinant enzyme, 37C, half-life is 8 h
738339
9
purified recombinant enzyme, 37C, half-life is 2 h, inactivation after 4 h
738339
10
purified recombinant enzyme, 37C, half-life is 1 h, inactivation after 3 h
738339
11
-
25C, 1 h, about 40% loss of activity
637321
12
-
25C, 1 h, about 60% loss of activity
637321
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
pH 7.5, 30 min, stable up to
37
-
thermostable up to
40
-
pH 7.5, 30 min, about 60% loss of activity
40 - 60
purified recombinant enzyme, pH 8.0, reasonably stable, maintaining 60% activity after 4 h at 40, and 50% activity after 30 min at 50C, and almost fully inactivated at 60C after 1 h
60
-
pH 7.5, 30 min, complete loss of activity
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
a specific cleavage by endogenous proteases at Lys280 of the 40000 Da enzyme. Cleavage results in the formation of two inactive fragments of 33000 Da and 7000 Da. The fragmentation is associated with a significant change of the enzyme from a tetrameric to trimeric form, and alterations in both secondary and native quarternary structures
-
expression of the native neuB gene product enzyme in E. coli results in a product that is prone to proteolysis during purification so the protein is tagged with a hexa-histidine tag at its N-terminus and rapidly purified
-
phosphoenolpyruvate has a major effect on the thermal stability of the wild-type enzyme requiring the C-terminal AFPL domain
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, purified His6-tagged recombinant enzyme, stable for only 3-4 weeks, the enzyme readily aggregated at room temperature
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cell harvesting by centrifugation, resuspension, lysing in high-pressure homogenizer, centrifugation, chromatographic procedures including ion-exchange and gel filtration steps, concentration
expression of the native neuB gene product enzyme in E. coli results in a product that is prone to proteolysis during purification so the protein is tagged with a hexa-histidine tag at its N-terminus and rapidly purified
-
Ni2+ affinity column chromatography
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recombinant His-tagged enzyme from Escherichia coli strain Rosetta 2 by ultrafiltration, nickel affinity chromatography, and gel filtration
recombinant His6-tagged enzyme from Escherichia coli strain Rosetta 2 (DE3) pLysS by nickel affinity chromatography and gel filtration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) Gold cells
-
expressed in Escherichia coli strain K12
expression in Escherichia coli (BL21 deltaDE3) with pCWori+ vector
expression of the neuB1 gene in Escherichia coli
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gene MvNeuB, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression of His6-tagged enzyme in Escherichia coli strain Rosetta 2 (DE3) pLysS
gene siaC, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis, recombinant expression of His-tagged enzyme in Escherichia coli strain Rosetta 2, subcloningin Escherichia coli strain DH5alpha
overexpression in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G133V
naturally occuring mutation of the enzyme causing impaired enzyme activity and the enzyme-deficiency phenotype
K131Q
naturally occuring mutation of the enzyme causing impaired enzyme activity and the enzyme-deficiency phenotype
P189L
naturally occuring mutation of the enzyme causing impaired enzyme activity and the enzyme-deficiency phenotype
Y188H
naturally occuring mutation of the enzyme causing impaired enzyme activity and the enzyme-deficiency phenotype
C182A
-
no activity
C182S
-
no activity
E134A
-
site-directed mutagenesis
E282A
-
site-directed mutagenesis
E282A/T285A
-
site-directed mutagenesis
T285A
-
site-directed mutagenesis
T285F
-
site-directed mutagenesis
E134A
-
site-directed mutagenesis
-
E282A
-
site-directed mutagenesis
-
E282A/T285A
-
site-directed mutagenesis
-
T285A
-
site-directed mutagenesis
-
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
Show AA Sequence (552 entries)
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