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Literature summary for 2.7.7.60 extracted from

  • 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 (2016), Sci. Rep., 6, 36379 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
gene ispD, recombinant expression of C-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3) Escherichia coli
gene ispD, recombinant expression of GST-tagged enzyme in Escherichia coli strain DH5alpha Bacillus subtilis

Crystallization (Commentary)

Crystallization (Comment) Organism
purified IspD and IspD complexed with CTP and Mg2+, hanging drop vapour diffusion method, for the apoenzyme, 8 mg/ml protein in 25 mM Tris-HCl, pH 8.0, and 0.1 M NaCl, is mixed with 0.1 M Tris (pH 8.5), 20% v/v PEG monomethyl ether 2000, for the complexed enzyme 8 mg/ml protein in 25 mM Tris-HCl, pH 8.0, and 0.1 M NaCl, is mixed with 0.2 M MgCl2, 0.1 M HEPES, pH 7.5, 25% v/v PEG 3350, 18°C, X-ray diffraction structure determination and analysis, molecular replacement using the monomer of IspD enzyme from Arabidopsis thaliana (AtIspD) (PDB ID 2yc3) as template Bacillus subtilis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information kinetic analysis Escherichia coli
additional information
-
additional information kinetic analysis Bacillus subtilis
0.1248
-
2-C-methyl-D-erythritol 4-phosphate recombinant detagged enzyme, pH 8.0, 30°C Bacillus subtilis
0.1328
-
CTP recombinant detagged enzyme, pH 8.0, 30°C Bacillus subtilis
0.2309
-
CTP recombinant detagged enzyme, pH 8.0, 30°C Escherichia coli
0.2915
-
2-C-methyl-D-erythritol 4-phosphate recombinant detagged enzyme, pH 8.0, 30°C Escherichia coli

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Escherichia coli
Mg2+ required Bacillus subtilis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
CTP + 2-C-methyl-D-erythritol 4-phosphate Escherichia coli
-
diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
-
?
CTP + 2-C-methyl-D-erythritol 4-phosphate Bacillus subtilis
-
diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
-
?
CTP + 2-C-methyl-D-erythritol 4-phosphate Bacillus subtilis 168
-
diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
-
?
CTP + 2-C-methyl-D-erythritol 4-phosphate Bacillus subtilis DSM 23778
-
diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
-
?

Organism

Organism UniProt Comment Textmining
Bacillus subtilis Q06755
-
-
Bacillus subtilis 168 Q06755
-
-
Bacillus subtilis DSM 23778 Q06755
-
-
Escherichia coli Q46893
-
-

Purification (Commentary)

Purification (Comment) Organism
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 Escherichia coli
recombinant GST-tagged enzyme from Escherichia coli strain DH5alpha by glutathione affinity chromatography, the GST tag is removed by Prescission Protease (PPase) enzyme, and followed by anion exchange chromatography and gel filtration, to 99% homogeneity Bacillus subtilis

Reaction

Reaction Comment Organism Reaction ID
CTP + 2-C-methyl-D-erythritol 4-phosphate = diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol conformational change of P-loop in the catalysis, concerted movements of the P-loop and L2-loop may be essential during the catalytic reaction, modeling Bacillus subtilis
CTP + 2-C-methyl-D-erythritol 4-phosphate = diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol IspD of Escherichia coli (EcIspD) follows stereochemical principles underlying the catalysis mechanism Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
CTP + 2-C-methyl-D-erythritol 4-phosphate
-
Escherichia coli diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
-
?
CTP + 2-C-methyl-D-erythritol 4-phosphate
-
Bacillus subtilis diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
-
?
CTP + 2-C-methyl-D-erythritol 4-phosphate
-
Bacillus subtilis 168 diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
-
?
CTP + 2-C-methyl-D-erythritol 4-phosphate
-
Bacillus subtilis DSM 23778 diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
-
?

Subunits

Subunits Comment Organism
dimer two crystal structures of BsIspD are determined in orthorhombic crystal form with space group P212121 and P21212, named apo form I and II, respectively. In the apo form I, two molecules in the asymmetric unit are related by 2fold sysmetry and form a dimer. In the apo form II, only one molecule is retained in the asymmetric unit, the functional dimer is formed by the sysmetry operation Bacillus subtilis
More the subunit structure of BsIspD is of a compact alpha/beta fold from which a long beta-meander extended. The core of the enzyme consists of a seven beta-sheets ( beta2, beta1, beta4, beta9, beta5, beta8, beta10 ) where all strands are parallel, apart from beta8 and beta2. The beta-meander lay between antiparallel strands beta6 and beta7 and made the major contribution to the dimer interface, and the lesser contribution came from the side-chain interactions of the residues on the alpha-helix fragment at the C-terminus. BsIspD structure, overview Bacillus subtilis

Synonyms

Synonyms Comment Organism
2-C-methyl-D-erythritol-4-phosphate cytidyltransferase
-
Escherichia coli
2-C-methyl-D-erythritol-4-phosphate cytidyltransferase
-
Bacillus subtilis
BsIspD
-
Bacillus subtilis
CDPME synthetase
-
Escherichia coli
CDPME synthetase
-
Bacillus subtilis
EcIspD
-
Escherichia coli
IspD
-
Escherichia coli
IspD
-
Bacillus subtilis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Escherichia coli
30
-
assay at Bacillus subtilis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
36.35
-
2-C-methyl-D-erythritol 4-phosphate recombinant detagged enzyme, pH 8.0, 30°C Bacillus subtilis
38.45
-
2-C-methyl-D-erythritol 4-phosphate recombinant detagged enzyme, pH 8.0, 30°C Escherichia coli
71.24
-
CTP recombinant detagged enzyme, pH 8.0, 30°C Bacillus subtilis
79.5
-
CTP recombinant detagged enzyme, pH 8.0, 30°C Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Escherichia coli
8
-
assay at Bacillus subtilis

General Information

General Information Comment Organism
additional information the concerted movements of the P-loop and loops close to the active site are essential in the reaction catalyzed by IspD. The intact P-loop is observed in the apo structure of IspD enzyme. The P-loop comprising residues 8-21 in the apo form I are refined satisfactorily, the B-factor of 30 A2 is almost the same as the average B-factor of all the protein atoms, this loop is highly conserved in the IspD enzymes. BsIspD structure, overview. The active site of BsIspD is covered by P-loop; hydrogen bonding interactions are formed between P-loop and adjacent residues. Upon CTP binding, conformational changes are observed on P-loop, L1-loop and L2-loop. The N-terminal half of the P-loop flips upward from active pocket and the C-terminal half flips down right, the active pocket is open to accommodate the CTP. A cleft is formed between L1-loop and P-loop, favoring for the binding of the cytosine base of CTP, at the same time, the residue Arg15 in the P-loop forms hydrogen bond to the residue Thr211 in L2-loop Bacillus subtilis
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 Escherichia coli
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 Bacillus subtilis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
131.9
-
2-C-methyl-D-erythritol 4-phosphate recombinant detagged enzyme, pH 8.0, 30°C Escherichia coli
291.3
-
2-C-methyl-D-erythritol 4-phosphate recombinant detagged enzyme, pH 8.0, 30°C Bacillus subtilis
344.3
-
CTP recombinant detagged enzyme, pH 8.0, 30°C Escherichia coli
536.5
-
CTP recombinant detagged enzyme, pH 8.0, 30°C Bacillus subtilis