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Information on EC 2.7.7.60 - 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase and Organism(s) Escherichia coli and UniProt Accession Q46893

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EC Tree
IUBMB Comments
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).
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This record set is specific for:
Escherichia coli
UNIPROT: Q46893
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The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
cytidylyltransferase, pfispd, 2-c-methyl-d-erythritol 4-phosphate cytidylyltransferase, aamct, mtispd, mep cytidylyltransferase, 2-c-methyl-d-erythritol-4-phosphate cytidyltransferase, 4-diphosphocytidyl-2c-methyl-d-erythritol synthase, cdp-me synthetase, hbcms, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2-C-methyl-D-erythritol-4-phosphate cytidyltransferase
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CDPME synthetase
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MEP cytidylyltransferase
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2-C-methyl erythritol 4-phosphate cytidylyltransferase
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-
-
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2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase
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-
-
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2-C-methylerythritol 4-cytidylyltransferase
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-
-
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4-diphosphocytidyl-2-C-methyl-D-erythritol synthetase
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-
-
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4-diphosphocytidyl-2-C-methylerythritol synthase
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-
-
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4-diphosphocytidyl-2C-methyl-D-erythritol synthase
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-
-
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CDP-ME synthetase
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-
-
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cytidylyltransferase, 2-C-methylerythritol 4-
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-
-
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diphosphocytidyl-methylerythritol synthetase
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MCT
-
-
-
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MEP cytidylyltransferase
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-
-
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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
CTP + 2-C-methyl-D-erythritol 4-phosphate = diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
show the reaction diagram
cytidyltransferase family. Active substrate site consists of a glycine-rich loop spanning Pro13-Arg20. Three-dimensional structure of enzyme is figured out
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
nucleotidyl group transfer
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-
-
-
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
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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-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
CTP + 2-C-methyl-D-erythritol-4-phosphate
diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
show the reaction diagram
additional information
?
-
-
lipoate-protein ligase attaches octanoate to the dehydrogenase subunit and sulfur insertion protein LipA then converts octanoate to lipoate. LipA acts on both octanoate and octanoyl-proteins
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
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
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essential enzyme in the mevalonate-independent pathway of isoprenoid biosynthesis
-
-
?
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
Co2+
-
catalytically active with
Mn2+
-
catalytically active with
additional information
-
Cu2+, Ni2+, Ca2+, Fe2+ or Zn2+ do not serve as cofactor
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
CTP
substrate inhibition
fosmidomycin
-
additional information
high-throughput screening to identify inhibitors of MEP cytidylyltransferase (IspD)
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.2915 - 131.5
2-C-methyl-D-erythritol 4-phosphate
0.131
4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
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0.003 - 6.6
CTP
0.061
2-C-methyl-D-erythritol 4-phosphate
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pH 7.5, 30°C
0.00314 - 0.032
2-C-methyl-D-erythritol-4-phosphate
0.061 - 0.131
CTP
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.4 - 52.7
2-C-methyl-D-erythritol 4-phosphate
0.26 - 79.5
CTP
16.8
2-C-methyl-D-erythritol-4-phosphate
-
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
131.9
2-C-methyl-D-erythritol 4-phosphate
recombinant detagged enzyme, pH 8.0, 30°C
344.3
CTP
recombinant detagged enzyme, pH 8.0, 30°C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
19.6
CTP
wild type enzyme, pH 6.5, 23°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23
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pure protein
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
assay at
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
the MEP cytidylyltransferase, or IspD catalyzes the second committed step of the methyl erythritol phosphate (MEP) pathway. The MEP pathway is essential for the production of isoprenoids
physiological function
2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (IspD) is an essential enzyme in the mevalonate-independent pathway of isoprenoid biosynthesis. This enzyme catalyzes 2-C-methyl-derythritol 4-phosphate (MEP) and cytosine triphosphate (CTP) to 4-diphosphocytidyl-2-C-methyl-derythritol (CDPME) and diphosphate
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25737
x * 25737 is predicted from sequence of c DNA
25700
26000
50000
-
gel filtration
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 25737 is predicted from sequence of c DNA
dimer
crystal structure analysis
?
-
x * 26000, SDS-PAGE
dimer
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
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vapor diffusion method
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vapor diffusion method at 4°C in hanging drops, crystals belong to space group C2
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D106A
no activity in crude cell free extract
D106E
strongly reduced but detectable activity
D106N
no activity in crude cell free extract
K214S
no activity in crude cell free extract
K27A
no activity in crude cell free extract
K27S
strongly reduced but detectable activity
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
K213S
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effective turnover rate is ca. 0.4% of the rate of wild type, retains capacity to form product with significantly reduced efficiency
K27A
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amino acid plays an essential role in catalysis because the effective turnover rate is ca. 0.025% of the rate of wild type enzyme
K27S
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amino acid plays an essential role in catalysis because the effective turnover rate is ca. 0.025% of the rate of wild type enzyme
additional information
-
mutants have been constructed that contain a defect in the pathway to convert 2-C-methyl-D-erythritol 4-phosphate in the unknown product, several genes are cloned to complement the defect of these blocked mutants
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant C-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, the His tag is removed by thrombin enzyme, and followed by anion exchange chromatography and gel filtration, to 99% homogeneity
recombinant His-tagged enzyme from Escherichia coli strain BL21 Codon Plus (DE3)-RIL by metal affinity chromatography and ultrafiltration
recombinant protein using His-tag
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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recombinant enzyme 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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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed as His-tag fusion protein in Escherichia coli BL21(DE3)
gene ispD, recombinant expression of C-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3)
gene ispD, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21 Codon Plus (DE3)-RIL
expressed as His-tag fusion protein in Escherichia coli M15
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expressed in Escherichia coli BL21(DE3)
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hyperexpression in Escherichia coli XL1-Blue
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overexpression of several genes that could complement the defect of the mutants
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
the methyl erythritol phosphate (MEP) pathway, including enzyme MEP cytidylyltransferase (IspD), represents an attractive series of targets for antibiotic design, considering each enzyme of the pathway is both essential and has no human homologues
biotechnology
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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
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due to the absence of the pathway in mammals enzyme provides a potential target for new antibiotics
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Cane, D.E.; Chow, C.; Lillo, A.; Kang, I.
Molecular cloning, expression and characterization of the first three genes in the mevalonate-independent isoprenoid pathway in Streptomyces coelicolor
Bioorg. Med. Chem.
9
1467-1477
2001
Escherichia coli, Streptomyces coelicolor, Streptomyces coelicolor CH999
Manually annotated by BRENDA team
Richard, S.B.; Bowman, M.E.; Kwiatkowski, W.; Kang, L.; Chow, C.; Lillo, A.M.; Cane, D.E.; Noel, J.P.
Structure of 4-diphosphocytidyl-2-C-methylerythritol synthetase involved in mevalonate-independent isoprenoid biosynthesis
Nature Struct. Biol.
8
641-648
2001
Escherichia coli, Mycobacterium tuberculosis, Plasmodium falciparum
Manually annotated by BRENDA team
Kemp, L.E.; Bond, C.S.; Hunter, W.N.
Crystallization and preliminary x-ray diffraction studies of recombinant Escherichia coli 4-diphosphocytidyl-2-C-methyl-D-erythritol synthetase
Acta Crystallogr. Sect. D
57
1189-1191
2001
Escherichia coli
Manually annotated by BRENDA team
Rohdich, F.; Wungsintaweekul, J.; Eisenreich, W.; Richter, G.; Schuhr, C.A.; Hecht, S.; Zenk, M.H.; Bacher, A.
Biosynthesis of terpenoids: 4-diphosphocytidyl-2C-methyl-D-erythritol synthase of Arabidopsis thaliana
Proc. Natl. Acad. Sci. USA
97
6451-6456
2000
Arabidopsis thaliana, Escherichia coli (Q46893), Escherichia coli
Manually annotated by BRENDA team
Kuzuyama, T.; Takagi, M.; Kaneda, K.; Dairi, T.; Seto, H.
Formation of 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol from 2-C-methyl-D-erythritol 4-phosphate by 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, a new enzyme in the nonmevalonate pathway
Tetrahedron Lett.
41
703-706
2000
Escherichia coli
-
Manually annotated by BRENDA team
Rohdich, F.; Wungsintaweekul, J.; Fellermeier, M.; Sagner, S.; Herz, S.; Kis, K.; Eisenreich, W.; Bacher, A.; Zenk, M.H.
Cytidine 5'-triphosphate-dependent biosynthesis of isoprenoids: YgbP protein of Escherichia coli catalyzes the formation of 4-diphosphocytidyl-2-C-methylerythritol
Proc. Natl. Acad. Sci. USA
96
11758-11763
1999
Arabidopsis thaliana, Escherichia coli, Escherichia coli DH5-alpha
Manually annotated by BRENDA team
Gabrielsen, M.; Bond, C.S.; Hallyburton, I.; Hecht, S.; Bacher, A.; Eisenreich, W.; Rohdich, F.; Hunter, W.N.
Hexameric assembly of the bifunctional methylerythritol 2,4-cyclodiphosphate synthase and protein-protein associations in the deoxy-xylulose-dependent pathway of isoprenoid precursor biosynthesis
J. Biol. Chem.
279
52753-52761
2004
Agrobacterium tumefaciens, Campylobacter jejuni, Escherichia coli (Q46893), Escherichia coli
Manually annotated by BRENDA team
Kemp, L.E.; Bond, C.S.; Hunter, W.N.
Structure of a tetragonal crystal form of Escherichia coli 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase
Acta Crystallogr. Sect. D
59
607-610
2003
Escherichia coli
Manually annotated by BRENDA team
Bernal, C.; Palacin, C.; Boronat, A.; Imperial, S.
A colorimetric assay for the determination of 4-diphosphocytidyl-2-C-methyl-D-erythritol 4-phosphate synthase activity
Anal. Biochem.
337
55-61
2005
Escherichia coli
Manually annotated by BRENDA team
Richard, S.B.; Lillo, A.M.; Tetzlaff, C.N.; Bowman, M.E.; Noel, J.P.; Cane, D.E.
Kinetic analysis of Escherichia coli 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase, wild type and mutants, reveals roles of active site amino acids
Biochemistry
43
12189-12197
2004
Escherichia coli (Q46893), Escherichia coli
Manually annotated by BRENDA team
Zhao, X.; Miller, J.R.; Jiang, Y.; Marletta, M.A.; Cronan, J.E.
Assembly of the covalent linkage between lipoic acid and its cognate enzymes
Chem. Biol.
10
1293-1302
2003
Escherichia coli
Manually annotated by BRENDA team
Illarionova, V.; Kaiser, J.; Ostrozhenkova, E.; Bacher, A.; Fischer, M.; Eisenreich, W.; Rohdich, F.
Nonmevalonate terpene biosynthesis enzymes as antiinfective drug targets: substrate synthesis and high-throughput screening methods
J. Org. Chem.
71
8824-8834
2006
Escherichia coli
Manually annotated by BRENDA team
Haymond, A.; Dowdy, T.; Johny, C.; Johnson, C.; Ball, H.; Dailey, A.; Schweibenz, B.; Villarroel, K.; Young, R.; Mantooth, C.J.; Patel, T.; Bases, J.; Dowd, C.S.; Couch, R.D.
A high-throughput screening campaign to identify inhibitors of DXP reductoisomerase (IspC) and MEP cytidylyltransferase (IspD)
Anal. Biochem.
542
63-75
2018
Escherichia coli (Q46893), Francisella tularensis subsp. novicida (A0Q5K1), Francisella tularensis subsp. novicida Utah 112 (A0Q5K1)
Manually annotated by BRENDA team
Jin, Y.; Liu, Z.; Li, Y.; Liu, W.; Tao, Y.; Wang, G.
A structural and functional study on the 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (IspD) from Bacillus subtilis
Sci. Rep.
6
36379
2016
Bacillus subtilis (Q06755), Bacillus subtilis, Escherichia coli (Q46893), Escherichia coli, Bacillus subtilis 168 (Q06755), Bacillus subtilis DSM 23778 (Q06755)
Manually annotated by BRENDA team