Information on EC 2.5.1.56 - N-acetylneuraminate synthase

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The enzyme appears in viruses and cellular organisms

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
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
CMP-N-acetylneuraminate biosynthesis II (bacteria)
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metabolism of amino sugars and derivatives
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Amino sugar and nucleotide sugar metabolism
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Metabolic pathways
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SYSTEMATIC NAME
IUBMB Comments
phosphoenolpyruvate:N-acetyl-D-mannosamine C-(1-carboxyvinyl)transferase (phosphate-hydrolysing, 2-carboxy-2-oxoethyl-forming)
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CAS REGISTRY NUMBER
COMMENTARY hide
37290-66-7
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain K1
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-
Manually annotated by BRENDA team
strain K1-M12
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-
Manually annotated by BRENDA team
strain 60E
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Manually annotated by BRENDA team
strain PRM102
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
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
?
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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
N-acetyl-D-mannosamine + phosphoenolpyruvate + H2O
?
show the reaction diagram
phosphoenolpyruvate + N-acetyl-D-mannosamine + H2O
N-acetylneuraminate + phosphate
show the reaction diagram
E0NCD4
-
-
-
?
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
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1 mM, 61% inhibition
Fe2+
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1 mM FeCl2, 40% inhibition
HgCl2
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at 0.01 mM 85% inhibition, at 0.1 mM complete inhibition
hydroxylamine
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1 mM, 23% inhibition
Iodine
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1 mM, 30% inhibition
K+
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1 mM KCl, 92% inhibition
Li+
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1 mM LiCl, 30% inhibition
N-bromosuccinimide
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1 mM, 24% inhibition
Na+
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1 mM NaCl, 20% inhibition
PCMB
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1 mM, complete inhibition
Phenylglyoxal
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N-acetylmannosamine or phosphoenolpyruvate protect
Semicarbazide
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1 mM, 20% inhibition
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
glutathione
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required for maximal activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5.6 - 17.6
N-acetyl-D-mannosamine
15
N-butanoyl-D-mannosamine
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pH 8.0, 37C
31.1
N-pentanoyl-D-mannosamine
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pH 8.0, 37C
5.5
N-propanoyl-D-mannosamine
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pH 8.0, 37C
0.04 - 7.3
phosphoenolpyruvate
additional information
additional information
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coupled assay to monitor enzyme kinetics
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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
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pH 8.0, 37C
0.8
N-pentanoyl-D-mannosamine
Campylobacter jejuni
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pH 8.0, 37C
11.8
N-propanoyl-D-mannosamine
Campylobacter jejuni
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pH 8.0, 37C
19
phosphoenolpyruvate
Campylobacter jejuni
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pH 8.0, 37C
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
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1.018
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pH 8.0, 37C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.3
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in Tris buffer
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 12
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pH 4.0: about 50% of maximal activity, pH 12.0: about 40% of maximal activity
6.5 - 10
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pH 6.5: about 40% of maximal activity, pH 10.0: about 60% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 60
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about 60% of maximal activity at 20C and at 60C
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38630
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calculated from amino acid sequence
106000
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gel filtration
135000
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recombinant protein containing a hexahistidine tag at the N-terminus, gel filtration
160000
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 43300, His-tagged recombinant enzyme, MALDI-TOF MS
monomer
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1 * 38629, calculated from amino acid sequence; 1 * 38649, ESI-MS
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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
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25C, 1 h, about 50% loss of activity
637321
5
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25C, 1 h, about 40% loss of activity
637321
6
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25C, 1 h, about 25% loss of activity
637321
7 - 10
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25C, 1 h, stable
637321
11
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25C, 1 h, about 40% loss of activity
637321
12
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25C, 1 h, about 60% loss of activity
637321
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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pH 7.5, 30 min, stable up to
37
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thermostable up to
40
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pH 7.5, 30 min, about 60% loss of activity
60
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pH 7.5, 30 min, complete loss of activity
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
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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
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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
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Ni2+ affinity column chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) Gold cells
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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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overexpression in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C182A
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no activity
C182S
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no activity
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
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