Information on EC 2.7.7.14 - ethanolamine-phosphate cytidylyltransferase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.7.7.14
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RECOMMENDED NAME
GeneOntology No.
ethanolamine-phosphate cytidylyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
CTP + ethanolamine phosphate = diphosphate + CDP-ethanolamine
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
nucleotidyl group transfer
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Glycerophospholipid metabolism
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Metabolic pathways
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phosphatidylethanolamine biosynthesis II
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Phosphonate and phosphinate metabolism
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plasmalogen biosynthesis
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phosphatidylethanolamine bioynthesis
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SYSTEMATIC NAME
IUBMB Comments
CTP:ethanolamine-phosphate cytidylyltransferase
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CAS REGISTRY NUMBER
COMMENTARY hide
9026-33-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
two evolutionary conserved splicing isoforms of Pcyt2, Pcyt2alpha and Pcyt2beta, and a third splicing isozyme Pcyt2gamma are encoded by a single Pcyt2 gene
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
two splicing isozymes Pcyt2alpha and Pcyt2beta
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
CTP + 2-aminoethylarsonic acid
diphosphate + cytidine-O-PO2-O-AsO2-CH2-CH2-NH3+
show the reaction diagram
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spontaneous hydrolysis to CMP and-O-AsO2--CH2-CH2-NH3+
?
CTP + 2-aminoethylphosphonate
?
show the reaction diagram
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-
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?
CTP + ethanolamine phosphate
CDP-ethanolamine + diphosphate
show the reaction diagram
CTP + ethanolamine phosphate
diphosphate + CDP-ethanolamine
show the reaction diagram
CTP + phosphocholine
diphosphate + CDP-choline
show the reaction diagram
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-
-
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?
CTP + phosphodimethylethanolamine
diphosphate + CDP-dimethylethanolamine
show the reaction diagram
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-
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?
CTP + phosphomonomethylethanolamine
diphosphate + CDP-methylethanolamine
show the reaction diagram
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-
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?
dCTP + ethanolamine phosphate
diphosphate + dCDP-ethanolamine
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 + ethanolamine phosphate
diphosphate + CDP-ethanolamine
show the reaction diagram
additional information
?
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Pcyt2 is the main regulatory enzyme in the CDP-ethanolamine pathwa
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
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activation, 10% of the activity with Mg2+
Co2+
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activation, 10% of the activity with Mg2+
additional information
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no activation by Ba2+, Zn2+, Cd2+, Ni2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-Aminoethylphosphonate
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inhibitory power stimulated by Mg2+; noncompetitive to phosphorylethanolamine
2-aminoethylphosphonate
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competitive to phosphorylethanolamine; inhibitory power stimulated by Mg2+
3-Aminopropylphosphonate
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competitive to phosphorylethanolamine; inhibitory power stimulated by Mg2+
aminoimidazole-4-carboxamide
CDP-ethanolamine
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CTP
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at concentrations exceeding that of Mg2+
diphosphate
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iodoacetamide
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phosphocholine
phosphodimethylethanolamine
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weak, competitive to phosphoethanolamine
phosphoethanolamine methyl-analogues
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phosphomonomethylethanolamine
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weak, competitive to phosphoethanolamine
Sphingosine/phosphatidylcholine vesicles
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inhibit cytosolic and purified enzyme
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additional information
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overactivation of the NMDA receptor in neurons inhibits the enzyme, membrane damage by NMDA receptor is preceeded by inhibition of phospholipid synthesis
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
AMP-activated kinase
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early growth response protein 1
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important transcriptional stimulator of gene expression
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paramethoxyamphetamine
phosphatidylglycerol
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slightly stimulating
Reducing agent
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additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3
2-Aminoethylarsonic acid
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approximate value, pH 7.8, 37°C
30
2-aminoethylphosphonate
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pH 5.5
0.01267 - 0.465
CTP
0.053 - 0.88
Ethanolamine phosphate
0.01209
ethanolamine-phosphate
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6.2
phosphocholine
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pH 7.8, 37°C
6.8
phosphodimethylethanolamine
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pH 7.8, 37°C
0.072
Phosphoethanolamine
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pH 7.8, 37°C
0.11
phosphomonomethylethanolamine
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pH 7.8, 37°C
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.015 - 1.15
CTP
0.016 - 4.1
Ethanolamine phosphate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
24
2-aminoethylphosphonate
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pH 5.5
52.9
phosphocholine
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pH 7.8, 37°C
6.8
phosphodimethylethanolamine
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pH 7.8, 37°C
7
phosphomonomethylethanolamine
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pH 7.8, 37°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1.13
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purified enzyme
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
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one sharp optimum at pH 7.8 and one with a lower maximal activity around pH 6.0
6.5
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2 optima: pH 6.5 and pH 8.0
8.5
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2 - 9
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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primary from brain cortical neurons
Manually annotated by BRENDA team
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the enzyme is downregulated in metastatic colon tumor
Manually annotated by BRENDA team
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minor part
Manually annotated by BRENDA team
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cortical
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
splice variant Pcyt2gamma
Manually annotated by BRENDA team
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inguinal and epididymal, upregulated in brown adipose tissue in comparison to white adipose tissue
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
34000
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x * 51000, isozyme Pcyt2alpha, x * 49000, isozyme Pcyt2beta, x * 34000, isozyme Pcyt2gamma
44623
1 * 44623, MALDI MS, analytical ultracentrifugation
49600
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x * 49600, SDS-PAGE
51000
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x * 51000, isozyme Pcyt2alpha, x * 49000, isozyme Pcyt2beta, x * 34000, isozyme Pcyt2gamma
90000
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myc-His-tagged Pcyt2beta, appears as two bands (110000 and 90000 Da), gel filtration
100000 - 120000
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gel filtration
108000
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myc-His-tagged Pcyt2alpha, gel filtration
110000
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myc-His-tagged Pcyt2beta, appears as two bands (110000 and 90000 Da), gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
1 * 44623, MALDI MS, analytical ultracentrifugation
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
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splice variant Pcyt2beta is specifically phosphorylated at the end of the first cytidylyltransferase domain. Splice variant Pcyt2alpha is phosphorylated within the alpha-specific motif that is spliced out in Pcyt2beta and on two protein kinase C consensus serine residues, Ser215 and Ser223. Single and double mutations of protein kinase C consensus sites reduce Pcyt2alpha phosphorylation, activity, and phosphatidylethanolamine synthesis by 50-90%. The phosphorylation and activity of endogenous Pcyt2 are dramatically increased with phorbol esters and reduced by specific protein kinase C inhibitors. In vitro translated Pcyt2 is phosphorylated by protein kinase Calpha, protein kinase CbetaI, and protein kinase CbetaII. The phosphorylated sites cluster within and flanking the central linker region that connects the two catalytic domains and is a regulatory segment not present in other cytidylyltransferases
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystallization by the sitting-drop vapour diffusion method using PEG 4000 as precipitant. The crystals diffract X-rays from a synchrotron-radiation source to 1.88 A resolution. The space group is assigned as primitive tetragonal, P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 66.3, c = 150.8. The crystals contain one ECT molecule in the asymmetric unit, with a solvent content of 43%
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
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limited stability below
642886
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
bovine serum albumin, 2% w/v, 1 mM CTP and 20 mM Mg2+, or 10% v/v glycerol stabilizes
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enzyme in crude postmicrosomal supernatant is quite stable towards freezing and thawing, but the highly purified enzyme loses 85-90% of its activity when frozen and thawed twice
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omission of DTT from buffers used in the later steps of purification results in severe loss of activity
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purified enzyme can be stabilized by the addition of 10% v/v glycerol or 2% bovine serum albumin
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quite stable towards freezing and thawing
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, stable for at least 4 weeks without significant loss of activity
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0-4°C, pH 7.5-9.0, in presence of DTT, stable for weeks
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
1162fold to homogeneity
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1430fold to homogeneity
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native enzyme from rat liver
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Ni ion affinity chromatography
Ni-NTA Superflow resin chromatography
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recombinant N-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cDNA encoding CTP:phosphoethanolamine cytidylyltransferase is transiently or stably transfected in CHO cells and a rat liver-derived cell line (McA-RH7777), resulting in a maximum of 26fold and 4fold increase in specific activity of CTP:phosphoethanolamine cytidylyltransferase respectively
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expressed in COS-7 cells
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expression in Escherichia coli as a fusion protein with maltose-binding protein
His-tagged version expressed in Escherichia coli BL21(DE3)
Pcyt2, DNA and amino acid sequence and promoter determination and analysis, three splicing isoforms of Pcyt2, alpha, beta, and gamma, encoded by a single Pcyt2 gene, genetic structures. Transcriptional regulation of Pcyt2, overview
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Pcyt2, two splicing isozymes Pcyt2alpha and Pcyt2beta, DNA and amino acid sequence and promoter determination and analysis, human Pcyt2, cDNA isolated from glioblastoma cells, is able to restore the synthesis of CDP-ethanolamine as well as the formation of PE in the enzyme-deficient yeast mutant. Transcriptional regulation of Pcyt2, overview
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Pcyt2, two splicing isozymes Pcyt2alpha and Pcyt2beta, DNA and amino acid sequence and promoter determination and analysis. Transcriptional regulation of Pcyt2, overview
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PfECT, sequence comparison, expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3)
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
25-hydroxycholesterol, an endogenous activator of liver X receptor, and the liver X receptor synthetic agonist TO901317 both significantly reduce the biosynthesis of phosphatidylethanolamine via the CDP-ethanolamine Kennedy pathway by inhibiting the promoter function and expression of Pcyt2 in human MCF-7 cells. The enzyme is downregulated in insulin-resistant muscle
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increases after serum starvation
induction of the enzyme by phorbol-12-myristate-13-acetate in hepatocytes. Liver X receptor, LXR, can modulate and activate promoter activity and transcription of Pcyt2. The enzyme is upregulated in obesity-resistant rats and by thiamine supplementation
liver X receptor, LXR, can modulate and activate promoter activity and transcription of Pcyt2. Pcyt2 is transcriptionally up-regulated by serum-deficiency induced differentiation of the skeletal muscle cells C2C12. The core mouse promoter (-111/+29)is dependent on binding of cEBP to an inverse CCAT box located at the position -82/-77 bp, ncreased amount of muscle-specific regulator, MyoD, reduced the content of Sp1 (binds to region -508/-378 bp), which, together with the decrease in ratio of Sp1 to Sp3, is responsible for the stimulation of transcription of Pcyt2 gene in differentiated C2C12 myotubes relative to undifferentiated myoblasts. The enzyme is upregulated in Sirtuin null mice and in adipose tissue of high-weight gainers
liver X receptor, LXR, can modulate and activate promoter activity and transcription of Pcyt2. Pcyt2 is upregulated in methotrexate-resistant HT-29 cells in comparison to a methotrexate-sensitive colon cancer cell line
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oxysterols, 24-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 24(S),25-epoxycholesterol, and mevalonolactate are partially responsible for the inhibition of Pcyt2 transcription. 25-Hydroxycholesterol, an endogenous activator of liver X receptor, and the liver X receptor synthetic agonist TO901317 both significantly reduce the biosynthesis of phosphatidylethanolamine via the CDP-ethanolamine Kennedy pathway by inhibiting the promoter function and expression of Pcyt2 in mouse embryonic fibroblasts. The enzyme is downregulated in sphingosine 1-phosphate lyase null mice and in livers of copper-transporting ATPase ATP7B null mice, downregulation in ATF2 null mice
significantly upregulated during muscle cell differentiation
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the important element for transcriptional control of Pcyt2 by 25-HC resides between -56 and -36 in NIH 3T3 cells. Nuclear factor-Y binds at C(-37)CAAT(-41) and Yin Yang1 binds at C(-42)AT(-40) in the Pcyt2 promoter. Nuclear factor-Y is involved in the inhibitory effects of 25-hydroxycholesterol on Pcyt2 transcription
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H226A
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mutation in histidine residue in the HxGH motif of the C-terminal CT domain. Decrease in activity
H229A
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mutation in histidine residue in the HxGH motif of the N-terminal CT domain. Activity similar to wild-type activity
H35A
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mutation in histidine residue in the HxGH motif of the N-terminal CT domain. Complete loss of activity
H38A
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mutation in histidine residue in the HxGH motif of the N-terminal CT domain. Strong decrease in activity
H244A
inactive C-domain mutant, presence of the mutant reduces splice variant Pcyt2alpha homodimerization and activity
H244Y
inactive C-domain mutant, presence of the mutant reduces splice variant Pcyt2alpha homodimerization and activity
H35A
inactive N-domain mutant, presence of the mutant reduces splice variant Pcyt2alpha homodimerization and activity
H35Y
inactive N-domain mutant, presence of the mutant reduces splice variant Pcyt2alpha homodimerization and activity
H146A
site-directed mutagenesis, the mutant is almost inactive
H422A
site-directed mutagenesis, the mutant shows increased kcat and Vmax with ethanolamine phosphate compared to the wild-type enzyme
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
given the absence of PE synthesis in red blood cells, PfECT represents a potential antimalarial target opening the way for a rational conception of bioactive compounds