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Literature summary for 2.7.7.60 extracted from

  • Shi, W.; Feng, J.; Zhang, M.; Lai, X.; Xu, S.; Zhang, X.; Wang, H.
    Biosynthesis of isoprenoids: characterization of a functionally active recombinant 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (IspD) from Mycobacterium tuberculosis H37Rv (2007), J. Biochem. Mol. Biol., 40, 911-920.
    View publication on PubMed
Search for more literature for 2.7.7.60

Application

Application Comment Organism
drug development insights in structure and function of MtIspD Mycobacterium tuberculosis

Cloned(Commentary)

Cloned (Comment) Organism
from genomic DNA in pET28a for expression with N-terminal hexa-His tag in Escherichia coli BL21 (DE3) for 12 h at 16°C Mycobacterium tuberculosis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.043
-
 2-C-methyl-D-erythritol 4-phosphate
-
 Mycobacterium tuberculosis
0.092
-
 CTP
-
 Mycobacterium tuberculosis

Metals/Ions

Metals/Ions Comment Organism Structure
Co2+
-
 Mycobacterium tuberculosis
divalent cation metal-dependent enzymatic activity Mycobacterium tuberculosis
Fe2+
-
 Mycobacterium tuberculosis
Mg2+ preferred Mycobacterium tuberculosis
Mn2+
-
 Mycobacterium tuberculosis

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
26110
-
 deduced from primary sequence Mycobacterium tuberculosis
26110
-
 SDS-polyacrylamide gel electrophoresis and mass spectrometry Mycobacterium tuberculosis
52000
-
 native polyacrylamide gel electrophoresis Mycobacterium tuberculosis

Organism

Organism UniProt Comment Textmining
Mycobacterium tuberculosis
-
 H37Rv
-
Mycobacterium tuberculosis H37Rv
-
 H37Rv
-

Purification (Commentary)

Purification (Comment) Organism
at 4°C, 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 Mycobacterium tuberculosis

Reaction

Reaction Comment Organism Reaction ID
CTP + 2-C-methyl-D-erythritol 4-phosphate = diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol third step in MEP pathway, isoprenoid biosynthesis Mycobacterium tuberculosis
a

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
7.8
-
-
 Mycobacterium tuberculosis

Storage Stability

Storage Stability Organism
-80°C, several months Mycobacterium tuberculosis
4°C, one month Mycobacterium tuberculosis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-C-methyl-D-erythritol 4-phosphate + ATP 13% activity compared to CTP as substrate, 30°C, 15 min, pH8 Mycobacterium tuberculosis diphosphate + 4-(adenine 5'-diphospho)-2-C-methyl-D-erythritol
-
 ?
2-C-methyl-D-erythritol 4-phosphate + ATP 13% activity compared to CTP as substrate, 30°C, 15 min, pH8 Mycobacterium tuberculosis H37Rv diphosphate + 4-(adenine 5'-diphospho)-2-C-methyl-D-erythritol
-
 ?
2-C-methyl-D-erythritol 4-phosphate + CTP 30°C, 15 min, pH8 Mycobacterium tuberculosis diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol coupled to hydrolysis of diphosphate to phosphate followed by complex formation with ammonium molybdate-malachite green (absorbance at 630 nm) ?
2-C-methyl-D-erythritol 4-phosphate + CTP 30°C, 15 min, pH8 Mycobacterium tuberculosis H37Rv diphosphate + 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol coupled to hydrolysis of diphosphate to phosphate followed by complex formation with ammonium molybdate-malachite green (absorbance at 630 nm) ?
additional information monofunctional enzyme Mycobacterium tuberculosis ?
-
 ?
additional information GTP, UTP and ITP are no substrates Mycobacterium tuberculosis ?
-
 ?
additional information monofunctional enzyme Mycobacterium tuberculosis H37Rv ?
-
 ?
additional information GTP, UTP and ITP are no substrates Mycobacterium tuberculosis H37Rv ?
-
 ?

Subunits

Subunits Comment Organism
homodimer
-
 Mycobacterium tuberculosis

Synonyms

Synonyms Comment Organism
2-C-methyl-D-erythritol 4-phosphate cytidyltransferase
-
 Mycobacterium tuberculosis
IspD
-
 Mycobacterium tuberculosis
MEP cytidyltransferase
-
 Mycobacterium tuberculosis
MtIspD ispD gene from Mycobacterium tuberculosis H37Rv, Rv3582c Mycobacterium tuberculosis

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
50
-
 sharp decrease in activity above Mycobacterium tuberculosis
80
-
 no remaining activity Mycobacterium tuberculosis

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
25 40 native-like tertiary structure, shapes of protein similar at 25°C, 30°C, and 40°C Mycobacterium tuberculosis
50 80 unstable at temperature above 50°C, estimated by circular dichroism at 25°C to 80°C (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 Mycobacterium tuberculosis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
additional information
-
 ATP 13% activity compared to CTP Mycobacterium tuberculosis
3.4
-
 CTP slightly lower than from bacterial and plant sources Mycobacterium tuberculosis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
-
 Mycobacterium tuberculosis