Information on EC 2.7.1.148 - 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase

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

EC NUMBER
COMMENTARY hide
2.7.1.148
-
RECOMMENDED NAME
GeneOntology No.
4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol = ADP + 2-phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phospho group transfer
-
-
-
-
Phosphorylation
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of antibiotics
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Biosynthesis of secondary metabolites
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isoprenoid biosynthesis
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Metabolic pathways
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methylerythritol phosphate pathway I
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methylerythritol phosphate pathway II
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Terpenoid backbone biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
ATP:4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol 2-phosphotransferase
The enzyme from Escherichia coli requires Mg2+ or Mn2+. Forms part of an alternative nonmevalonate pathway for terpenoid biosynthesis (for diagram, click here).
CAS REGISTRY NUMBER
COMMENTARY hide
263016-77-9
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
UniProt
Manually annotated by BRENDA team
Escherichia coli DH5-alpha
-
-
-
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
tomato
Uniprot
Manually annotated by BRENDA team
strain HB8
-
-
Manually annotated by BRENDA team
Vitis vinifera x Vitis riparia
-
-
-
Manually annotated by BRENDA team
Vitis vinifera x Vitis vinifera
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
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enzyme catalyses the fourth reaction step of the 2C-methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of isopentenyl pyrophosphate and its isomer dimethylallyl pyrophosphate
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol + ATP
2-phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol + ADP
show the reaction diagram
-
-
-
ir
4-diphosphocytidyl-2C-methyl-D-erythritol + ATP
4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate + ADP
show the reaction diagram
non-mevalonate pathway of isoprenoid precursor synthesis
-
-
ir
ATP + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
ADP + 2-phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
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
4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol + ATP
2-phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol + ADP
show the reaction diagram
A6XAA8, A6XAA9
-
-
-
ir
4-diphosphocytidyl-2C-methyl-D-erythritol + ATP
4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate + ADP
show the reaction diagram
O67060
non-mevalonate pathway of isoprenoid precursor synthesis
-
-
ir
ATP + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
ADP + 2-phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
show the reaction diagram
additional information
?
-
-
in Agrobacterium tumefaciens the ispD and ispF genes are fused to encode a bifunctional enzyme that catalyzes the first (synthesis of 4-diphosphocytidyl-2-C-methyl D-erythritol) and third (synthesis of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate) steps of isoprenoid biosynthetic pathway. Enzyme-generated intermediates are not transferred from the IspD active site in IspDF to the active site of IspE or from the active site in IspE to the active site of the IspF module of IspDF
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-
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
activity rate 8% compared to Mg2+
Co2+
activity rate 52% compared to Mg2+
Cu2+
activity rate 40% compared to Mg2+
Fe2+
activity rate 16% compared to Mg2+
Mn2+
activity rate 94% compared to Mg2+
Ni2+
activity rate 11% compared to Mg2+
Zn2+
activity rate 14% compared to Mg2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(4E)-3-methyl-4-[(5-phenylfuran-2-yl)methylidene]-1,2-oxazol-5(4H)-one
(4Z)-4-(furan-2-ylmethylidene)-3-phenyl-1,2-oxazol-5(4H)-one
(4Z)-4-[(5-methylfuran-2-yl)methylidene]-3-phenyl-1,2-oxazol-5(4H)-one
6-(benzylsulfanyl)-2-(2-hydroxyphenyl)-4-oxo-3,4-dihydro-2H-1,3-thiazine-5-carbonitrile
ethyl [4-amino-2-oxo-5-(3-[[(2,2,2-trifluoroethyl)sulfonyl]amino]prop-1-en-1-yl)pyrimidin-1(2H)-yl]acetate
-
mixed type
N-[3-(4-amino-1-benzyl-2-oxo-1,2-dihydropyrimidin-5-yl)prop-2-en-1-yl]ethanesulfonamide
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mixed-type
N-[3-[4-amino-2-oxo-1-(1H-pyrazol-5-ylmethyl)-1,2-dihydropyrimidin-5-yl]prop-2-en-1-yl]ethanesulfonamide
-
competitive
N-[3-[4-amino-2-oxo-1-(tetrahydro-2-thienyl)-1,2-dihydropyrimidin-5-yl]prop-2-en-1-yl]-1,1,1-trifluoromethanesulfonamide
-
competitive
N-[3-[4-amino-2-oxo-1-(tetrahydro-2-thienyl)-1,2-dihydropyrimidin-5-yl]prop-2-en-1-yl]-2,2,2-trifluoroethanesulfonamide
-
competitive
N-[3-[4-amino-2-oxo-1-(tetrahydro-2-thienyl)-1,2-dihydropyrimidin-5-yl]prop-2-en-1-yl]benzenesulfonamide
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competitive
N-[3-[4-amino-2-oxo-1-(tetrahydro-2-thienyl)-1,2-dihydropyrimidin-5-yl]prop-2-en-1-yl]cyclopropanesulfonamide
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competitive
N-[3-[4-amino-2-oxo-1-(tetrahydro-2-thienyl)-1,2-dihydropyrimidin-5-yl]prop-2-en-1-yl]ethanesulfonamide
-
competitive
N-[3-[4-amino-2-oxo-1-(tetrahydro-2-thienyl)-1,2-dihydropyrimidin-5-yl]prop-2-en-1-yl]methanesulfonamide
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competitive
N-[3-[4-amino-2-oxo-1-(tetrahydro-2-thienyl)-1,2-dihydropyrimidin-5-yl]prop-2-en-1-yl]propane-1-sulfonamide
-
mixed-type
N-[3-[4-amino-2-oxo-1-(tetrahydrofuran-2-ylmethyl)-1,2-dihydropyrimidin-5-yl]prop-2-en-1-yl]-2,2,2-trifluoroethanesulfonamide
-
competitive
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
light
Selective increase in the transcript abundance of GbCMK2 in the radicles by induction of light.
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methyl jasmonate
Selective increase in the transcript abundance of GbCMK2 in adicles.
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.2
4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
-
pH not specified in the publication, 22C
0.121
4-diphosphocytidyl-2C-methyl-D-erythritol
-
0.02 - 0.222
ATP
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0042
ethyl [4-amino-2-oxo-5-(3-[[(2,2,2-trifluoroethyl)sulfonyl]amino]prop-1-en-1-yl)pyrimidin-1(2H)-yl]acetate
-
competitive inhibition constant
0.0037
N-[3-(4-amino-1-benzyl-2-oxo-1,2-dihydropyrimidin-5-yl)prop-2-en-1-yl]ethanesulfonamide
-
competitive inhibition constant
0.0016
N-[3-[4-amino-2-oxo-1-(1H-pyrazol-5-ylmethyl)-1,2-dihydropyrimidin-5-yl]prop-2-en-1-yl]ethanesulfonamide
-
competitive inhibition constant
0.0012
N-[3-[4-amino-2-oxo-1-(tetrahydro-2-thienyl)-1,2-dihydropyrimidin-5-yl]prop-2-en-1-yl]-1,1,1-trifluoromethanesulfonamide
-
competitive inhibition constant
0.00036
N-[3-[4-amino-2-oxo-1-(tetrahydro-2-thienyl)-1,2-dihydropyrimidin-5-yl]prop-2-en-1-yl]-2,2,2-trifluoroethanesulfonamide
-
competitive inhibition constant
0.0163
N-[3-[4-amino-2-oxo-1-(tetrahydro-2-thienyl)-1,2-dihydropyrimidin-5-yl]prop-2-en-1-yl]benzenesulfonamide
-
competitive inhibition constant
0.00029
N-[3-[4-amino-2-oxo-1-(tetrahydro-2-thienyl)-1,2-dihydropyrimidin-5-yl]prop-2-en-1-yl]cyclopropanesulfonamide
-
competitive inhibition constant
0.00064
N-[3-[4-amino-2-oxo-1-(tetrahydro-2-thienyl)-1,2-dihydropyrimidin-5-yl]prop-2-en-1-yl]ethanesulfonamide
-
competitive inhibition constant
0.0026
N-[3-[4-amino-2-oxo-1-(tetrahydro-2-thienyl)-1,2-dihydropyrimidin-5-yl]prop-2-en-1-yl]methanesulfonamide
-
competitive inhibition constant
0.0082
N-[3-[4-amino-2-oxo-1-(tetrahydro-2-thienyl)-1,2-dihydropyrimidin-5-yl]prop-2-en-1-yl]propane-1-sulfonamide
-
competitive inhibition constant
0.0323
N-[3-[4-amino-2-oxo-1-(tetrahydrofuran-2-ylmethyl)-1,2-dihydropyrimidin-5-yl]prop-2-en-1-yl]-2,2,2-trifluoroethanesulfonamide
-
competitive inhibition constant
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0055 - 0.006
(4E)-3-methyl-4-[(5-phenylfuran-2-yl)methylidene]-1,2-oxazol-5(4H)-one
0.007 - 0.012
(4Z)-4-(furan-2-ylmethylidene)-3-phenyl-1,2-oxazol-5(4H)-one
0.008 - 0.013
(4Z)-4-[(5-methylfuran-2-yl)methylidene]-3-phenyl-1,2-oxazol-5(4H)-one
0.009 - 0.018
6-(benzylsulfanyl)-2-(2-hydroxyphenyl)-4-oxo-3,4-dihydro-2H-1,3-thiazine-5-carbonitrile
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1.5
in presence of 2 mM Mg2+ or Mn2+
33
purified recombinant protein
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35 - 37
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assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.1
theoretical value, predicted
5.2
theoretical value, predicted
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
cloned in Escherichia coli
Manually annotated by BRENDA team
5 embryos are pooled to prepare a RNA sample, used for complementation assay; 5 embryos are pooled to prepare a RNA sample, used for complementation assay
Manually annotated by BRENDA team
5 embryos are pooled to prepare a RNA sample, used for complementation assay; 5 embryos are pooled to prepare a RNA sample, used for complementation assay
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
isoenzyme contains chloroplast transit peptide sequence; isoenzyme contains chloroplast transit peptide sequence
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Escherichia coli (strain K12)
Escherichia coli O6:H1 (strain CFT073 / ATCC 700928 / UPEC)
Mycobacterium abscessus (strain ATCC 19977 / DSM 44196 / CIP 104536 / JCM 13569 / NCTC 13031 / TMC 1543)
Mycobacterium abscessus (strain ATCC 19977 / DSM 44196 / CIP 104536 / JCM 13569 / NCTC 13031 / TMC 1543)
Mycobacterium abscessus (strain ATCC 19977 / DSM 44196 / CIP 104536 / JCM 13569 / NCTC 13031 / TMC 1543)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30000
by gel-filtration chromatography
36400
calculated from cDNA sequence
39200
theoretical value, predicted
39900
theoretical value, predicted
62000
-
determined by MALDI-TOF-MS
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
-
determined by gel filtration, asymmetric unit consists of 2 subunits A and B which assemble with C2 symmetry to form an extended homodimer, but only 4% of the total surface area of the 2 monomers is involved in dimer formation
monomer
Gel-filtration and analytical ultracentrifugation indicate that AaIspE is a monomer in solution
additional information
-
the enzyme subunit displays the alpha/beta fold characteristic of the galactose kinase/homoserine kinase/mevalonate kinase phosphomevalonate kinase superfamily, arranged into cofactor and substrate-binding domains with the catalytic center positioned in a deep cleft between domains, quarternary structure, overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
AaIspE is crystallized by vapor diffusion. Ordered and reproducible crystals of AaIspE are obtained and multiple-wavelength anomalous dispersion methods are applied to obtain the initial phases. 3 medium-resolution complex crystal structures are determined. The crystals are isomorphous with two molecules in the asymmetric unit.
in complex with ADP, to 2.0 A resolution, and comparison with a monoclinic crystal form of a ternary complex of Escherichia coli 4-diphosphocytidyl-2C-methyl-D-erythritol kinase also with two molecules in the asymmetric unit. The molecular packing is different in the two forms. In the asymmetric unit of the triclinic crystal form the substrate-binding sites are occluded by structural elements of the partner, suggesting that the triclinic dimer is an artefact of the crystal lattice. The surface area of interaction in the triclinic form is almost double that observed in the monoclinic form
purified recombinant enzyme in ternary complex with substrate and non-hydrolyzable ATP analogue adenosine 5'-[beta,gamma-imino]triphosphate, i.e. AMP-PNP, 25 mg/ml protein in 50 mM Tris-HCl, pH 7.7, 50 mM NaCl, 3 mM AMP-PNP, and 2 mM substrate 4-(cytidine 5'-phosphate)-2-C-methyl-D-erythritol, hanging drop vapour diffusion method, 0.001 ml protein solution mixed with equal volume of reservoir solution containing 20% polyethylene glycol 8000, 0.2 M magnesium acetate, 0.1 M sodium cacodylate, pH 6.5, and 0.0002 ml of 0.25 sulfo-betaine, cryoprotection at -173C by 20% glycerol, X-ray diffraction structure determination and analysis at 2.0 A resolution
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structure-activity relationship studies with inhibitors 6-(benzylsulfanyl)-2-(2-hydroxyphenyl)-4-oxo-3,4-dihydro-2H-1,3-thiazine-5-carbonitrile and (4E)-3-methyl-4-[(5-phenylfuran-2-yl)methylidene]-1,2-oxazol-5(4H)-one
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the structure is resolved by X-ray diffraction at a resolution of 2.01 A
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free enzyme, in complex with substrates 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol, ADP and with adenyl imidodiphosphate, to 1.0, 1.54, 1.0, and 1.0 A resolution, respectively. The structures present a characteristic galactose/homoserine/mevalonate/phosphomevalonate kinase superfamily alpha/beta-fold with a catalytic center located in a cleft between two domains and display clear substrate and ATP binding pockets
purified recombinant selenomethionine-labeled enzyme, hanging drop vapour diffusion method, 20C, 0.002 ml protein solution containing 2.2 mg/ml protein, 20 ml Tris-HCl, pH 8.0, and 50 mM NaCl, are mixed with 0.001 ml mother liquor containing 33 mM Tris-HCl, pH 8.5, 67 mM sodium acetate, 13% isopropanol, 8% butanol, and 13% PEG 4000, cryoprotection at -173C in 30% glycerol, X-ray diffraction structure determination and analysis at 1.7 A resolution
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structure-activity relationship studies with inhibitors 6-(benzylsulfanyl)-2-(2-hydroxyphenyl)-4-oxo-3,4-dihydro-2H-1,3-thiazine-5-carbonitrile and (4E)-3-methyl-4-[(5-phenylfuran-2-yl)methylidene]-1,2-oxazol-5(4H)-one
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Hi-trap chelating columns are used for the purification of recombinant 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase
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recombinant enzyme
recombinant His-tagged enyzme from strain BL21(DE3) to homogeneity by nickel affinity and anion exchange chromatography
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recombinant His6-tagged enzyme by nickel affinity chromatography
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recombinant selenomethionine-labeled enzyme from Escherichia coli strain B834(DE3) by ammonium sulfate fractionation, hydrophobic interaction and heparin affinity chromatography, gel filtration, and ultrafiltration
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The gene is preceded by a His6 tag to enable purification of the recombinant protein via metal-chelating affinity chromatography. The polyhistidine tag is removed by thrombin-mediated proteolysis, followed by purification with anion-exchange chromatography. Purity of the sample is assessed by SDS-PAGE and MALDI-TOF MS.
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gene ispE, expression of the His-tagged in strain BL21(DE3)
-
gene ychB, expression of the selenomethionine-labeled enzyme in Escherichia coli strain B834(DE3)
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gene ychB, overexpression of His6-tagged enzyme
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overexpression in Escherichia coli
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putative catalytic domain, residues 81-401
The putative ispE gene from Aquifex aeolicus is placed under the control of the T7 promoter and lac operator in the hyperexpression plasmid pET-15b, and heat transformed into Escherichia coli BL21 (DE3).
The two CMK sequences encoding the mature CMK proteins are amplified with their respective primer pairs and cloned to the pMW118 vector to derive the pMW-CMK1 and the pMW-CMK2 plasmids. NMW29, an Escherichia coli ychB mutant harboring the pTMV20KM plasmid. The strain is transformed with the pMW-CMK1 and the pMW-CMK2 plasmids and selected on the LB medium without mevalonate.; The two CMK sequences encoding the mature CMK proteins are amplified with their respective primer pairs and cloned to the pMW118 vector to derive the pMW-CMK1 and the pMW-CMK2 plasmids. NMW29, an Escherichia coli ychB mutant harboring the pTMV20KM plasmid. The strain is transformed with the pMW-CMK1 and the pMW-CMK2 plasmids and selected on the LB medium without mevalonate.
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D152A
-
inactive mutant enzyme
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
medicine
-
potential target for antimalarial therapy
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