Information on EC 2.7.7.60 - 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase

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

EC NUMBER
COMMENTARY hide
2.7.7.60
-
RECOMMENDED NAME
GeneOntology No.
2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
CTP + 2-C-methyl-D-erythritol 4-phosphate = diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
nucleotidyl group transfer
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of antibiotics
-
-
Biosynthesis of secondary metabolites
-
-
isoprenoid biosynthesis
-
-
Metabolic pathways
-
-
methylerythritol phosphate pathway I
-
-
methylerythritol phosphate pathway II
-
-
Terpenoid backbone biosynthesis
-
-
SYSTEMATIC NAME
IUBMB Comments
CTP:2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase
The enzyme from Escherichia coli requires Mg2+ or Mn2+. ATP or UTP can replace CTP, but both are less effective. GTP and TTP are not substrates. Forms part of an alternative nonmevalonate pathway for terpenoid biosynthesis (for diagram, click here).
CAS REGISTRY NUMBER
COMMENTARY hide
251990-59-7
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Escherichia coli DH5-alpha
DH5alpha
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
CH999
-
-
Manually annotated by BRENDA team
CH999
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
2-C-methyl-D-erythritol 4-phosphate + ATP
diphosphate + 4-(adenine 5'-diphospho)-2-C-methyl-D-erythritol
show the reaction diagram
2-C-methyl-D-erythritol 4-phosphate + CTP
diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
show the reaction diagram
CTP + 2-C-methyl-D-erythritol 4-phosphate
diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
show the reaction diagram
CTP + 2-C-methyl-D-erythritol-4-phosphate
diphosphate + 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
CTP + 2-C-methyl-D-erythritol 4-phosphate
diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
show the reaction diagram
CTP + 2-C-methyl-D-erythritol-4-phosphate
diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Divalent cation
-
metal-dependent enzymatic activity
additional information
-
Cu2+, Ni2+, Ca2+, Fe2+ or Zn2+ do not serve as cofactor
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
CTP
-
substrate inhibition
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.043 - 131.5
2-C-methyl-D-erythritol 4-phosphate
0.00314 - 3.26
2-C-methyl-D-erythritol-4-phosphate
0.131 - 0.5
4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
0.003 - 6.6
CTP
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.4 - 52.7
2-C-methyl-D-erythritol 4-phosphate
1.4 - 16.8
2-C-methyl-D-erythritol-4-phosphate
0.26 - 54.1
CTP
additional information
ATP
Mycobacterium tuberculosis
-
13% activity compared to CTP
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5.66
2-C-methyl-D-erythritol 4-phosphate
Francisella tularensis
-
pH 8.0, 37C
2166
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
19.6
CTP
-
wild type enzyme, pH 6.5, 23C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00335
-
assay with D-1-deoxyxylulose-5-phosphate or 2-C-methyl-D-erythritol-4-phosphate
23
-
pure protein
33
-
cell extract
67
-
pure protein
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
-
sharp decrease in activity above
80
-
no remaining activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9.1
calculated
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
transcription level of enzyme remains constant for at least one month
Manually annotated by BRENDA team
embryonic root, transcription level of enzyme remains constant for at least one month
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Anaerococcus prevotii (strain ATCC 9321 / DSM 20548 / JCM 6508 / PC1)
Burkholderia thailandensis (strain E264 / ATCC 700388 / DSM 13276 / CIP 106301)
Burkholderia thailandensis (strain E264 / ATCC 700388 / DSM 13276 / CIP 106301)
Campylobacter jejuni subsp. jejuni serotype O:2 (strain ATCC 700819 / NCTC 11168)
Campylobacter jejuni subsp. jejuni serotype O:2 (strain ATCC 700819 / NCTC 11168)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Francisella tularensis subsp. holarctica (strain LVS)
Listeria monocytogenes serotype 4b (strain F2365)
Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155)
Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Neisseria gonorrhoeae (strain ATCC 700825 / FA 1090)
Neisseria gonorrhoeae (strain ATCC 700825 / FA 1090)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25900
-
predicted from cDNA
26110
-
deduced from primary sequence; SDS-polyacrylamide gel electrophoresis and mass spectrometry
50000
-
gel filtration
52000
-
native polyacrylamide gel electrophoresis
250000
-
analytical ultracentrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer
homodimer
monomer
-
sedimentation velocity experiments, both wild-type and mutant D152A
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
proteolytic modification
sequence contains a N-terminal chloroplast transit peptide of 88 amino acids
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method
-
catalytic domain of recombinant enzyme, structure contains a cytidine monophosphate bound in the active site suggesting a mechanism for feedback regulation of enzyme activity
hanging drop vapor diffusion method
hanging-drop vapour-diffusion using paoyethylene glycol as precipitant, elongated tetragonal prismatic crystals of 0.1 * 0.1 * 0.5 mm, space group P41212
-
vapor diffusion method
-
vapor diffusion method at 4C in hanging drops, crystals belong to space group C2
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 40
-
native-like tertiary structure, shapes of protein similar at 25C, 30C, and 40C
50 - 80
-
unstable at temperature above 50C, estimated by circular dichroism at 25C to 80C (far-UV and near-UV spectra) and after 10 min equilibration, partly integer secondary structure, occurrence of thermally-induced unfolding correlated with catalytic activity loss
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80C, several months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
at 4C, from bacterial lysate by Ni-NTA metal affinity chromatography (elution with 200 mM imidazole), dialysis, concentration by ultrafiltration, yield of 2.8 mg/l culture
-
metal-chelating Hitrap clumn preloaded with Ni2+, Q-Sepharose anion-exchange chromatography to remove previously added thrombin
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Ni2+-nitrilotriacetic acid column
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Q-Sepharose, Phenyl-Sepharose, Superdex HiLoad
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recombinant enzyme using His-tag
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recombinant protein using His-tag
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Sepharose Q FF column, Phenyl Sepharose, Cibacron blue 3GA column, Red Sepharose CL-6B column, Source 15 Q column
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Source 15 Q and Superdex 200 column
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using metal affinity chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed as His-tag fusion protein in Escherichia coli BL21(DE3)
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expressed as His-tag fusion protein in Escherichia coli M15
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expressed in Escherichia coli as a His-tagged fusion protein
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expressed in Escherichia coli BL21(DE3)
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expressed in Escherichia coli BL21(DE3)-RP
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expressed in Escherichia coli M15(pREP4)
-
from genomic DNA in pET28a for expression with N-terminal hexa-His tag in Escherichia coli BL21 (DE3) for 12 h at 16C
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hyperexpression in Escherichia coli XL1-Blue
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into the vector pT7Blue for sequencing, a cDNA library is constructed in a PDONR222 vector; into the vector pT7Blue for sequencing, a cDNA library is constructed in a PDONR222 vector
overexpression of several genes that could complement the defect of the mutants
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D152A
-
catalytically inactive
D106A
-
no activity in crude cell free extract
D106E
-
strongly reduced but detectable activity
D106N
-
no activity in crude cell free extract
K213S
-
effective turnover rate is ca. 0.4% of the rate of wild type, retains capacity to form product with significantly reduced efficiency
K214S
-
no activity in crude cell free extract
R109A
-
strongly reduced but detectable activity
R19A
-
no effect on activity
R19K
-
no effect on activity
R20A
-
no effect on activity
R20K
-
reduced activity
T140V
-
strongly reduced but detectable activity
T165V
-
no activity in crude cell free extract
T141D
-
mutant shows only 25% of wild-type activity
T141E
-
mutant catalytically inactive
T141D
-
mutant shows only 25% of wild-type activity
-
T141E
-
mutant catalytically inactive
-
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
-
high-throughput methods for the screening of 2C-methyl-D-erythritol synthase IspC protein, 4-diphosphocytidyl-2C-methyl-D-erythritol synthase IspD protein, 4-diphosphocytidyl-2Cmethyl-D-erythritol kinase IspE protein, and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF protein, against large compound libraries. Assays use up to three auxiliary enzymes monitored at 340 and all robust
drug development
medicine
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