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Literature summary 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

Application

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

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.7.60 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.7.7.60 0.043
-
 2-C-methyl-D-erythritol 4-phosphate
-
 Mycobacterium tuberculosis
2.7.7.60 0.092
-
 CTP
-
 Mycobacterium tuberculosis

Metals/Ions

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

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.7.7.60 26110
-
 deduced from primary sequence Mycobacterium tuberculosis
2.7.7.60 26110
-
 SDS-polyacrylamide gel electrophoresis and mass spectrometry Mycobacterium tuberculosis
2.7.7.60 52000
-
 native polyacrylamide gel electrophoresis Mycobacterium tuberculosis

Organism

EC Number Organism UniProt Comment Textmining
2.7.7.60 Mycobacterium tuberculosis
-
 H37Rv
-
2.7.7.60 Mycobacterium tuberculosis H37Rv
-
 H37Rv
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.7.60 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

EC Number Reaction Comment Organism Reaction ID
2.7.7.60 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]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.7.7.60 7.8
-
-
 Mycobacterium tuberculosis

Storage Stability

EC Number Storage Stability Organism
2.7.7.60 -80°C, several months Mycobacterium tuberculosis
2.7.7.60 4°C, one month Mycobacterium tuberculosis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.7.60 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
-
 ?
2.7.7.60 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
-
 ?
2.7.7.60 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) ?
2.7.7.60 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) ?
2.7.7.60 additional information monofunctional enzyme Mycobacterium tuberculosis ?
-
 ?
2.7.7.60 additional information GTP, UTP and ITP are no substrates Mycobacterium tuberculosis ?
-
 ?
2.7.7.60 additional information monofunctional enzyme Mycobacterium tuberculosis H37Rv ?
-
 ?
2.7.7.60 additional information GTP, UTP and ITP are no substrates Mycobacterium tuberculosis H37Rv ?
-
 ?

Subunits

EC Number Subunits Comment Organism
2.7.7.60 homodimer
-
 Mycobacterium tuberculosis

Synonyms

EC Number Synonyms Comment Organism
2.7.7.60 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase
-
 Mycobacterium tuberculosis
2.7.7.60 IspD
-
 Mycobacterium tuberculosis
2.7.7.60 MEP cytidyltransferase
-
 Mycobacterium tuberculosis
2.7.7.60 MtIspD ispD gene from Mycobacterium tuberculosis H37Rv, Rv3582c Mycobacterium tuberculosis

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
2.7.7.60 50
-
 sharp decrease in activity above Mycobacterium tuberculosis
2.7.7.60 80
-
 no remaining activity Mycobacterium tuberculosis

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
2.7.7.60 25 40 native-like tertiary structure, shapes of protein similar at 25°C, 30°C, and 40°C Mycobacterium tuberculosis
2.7.7.60 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]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.7.7.60 additional information
-
 ATP 13% activity compared to CTP Mycobacterium tuberculosis
2.7.7.60 3.4
-
 CTP slightly lower than from bacterial and plant sources Mycobacterium tuberculosis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.7.7.60 8
-
-
 Mycobacterium tuberculosis