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Literature summary extracted from

  • Herrscher, V.; Witjaksono, C.; Buchotte, M.; Ferret, C.; Massicot, F.; Vasse, J.L.; Borel, F.; Behr, J.B.; Seemann, M.
    Irreversible inhibition of IspG, a target for the development of new antimicrobials, by a 2-vinyl analogue of its MEcPP substrate (2022), Chemistry, 28, e202200241.
    View publication on PubMed

Application

EC Number Application Comment Organism
1.17.7.3 drug development the penultimate step of the methylerythritol phosphate (MEP) pathway is a validated target for drug development. A 2-vinyl analogue of substrate 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate (MEcPP) is designed to generate conjugated species during enzyme catalysis, with the aim of providing reactive centers to be covalently trapped by neighboring amino acid residues. The synthesized substrate analogue displays irreversible inhibition towards IspG Escherichia coli

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.17.7.3 gene ispG, recombinant overexpression of His-tagged enzyme in Escherichia coli strain XL1-blue Escherichia coli

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.17.7.3 2-C-vinyl-D-erythritol-2,4-cyclodiphosphate VEcPP, irreversible inhibitor, synthesis, overview. The 2-vinyl analogue of substrate 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate (MEcPP) is designed to generate conjugated species during enzyme catalysis, with the aim of providing reactive centers to be covalently trapped by neighboring amino acid residues. The synthesized substrate analogue displays irreversible inhibition towards IspG. The electron transfer occurs prior to inhibition. Inhibition method evaluation and optimization, overview. A sample of IspG preincubated with the inhibitior in the absence of the reduction system retaines its activity after removing the inhibitor from the reaction mixture showing that the flavodoxin/flavodoxin reductase/NADPH system is required during preincubation to achieve IspG inhibition, suggesting that the inhibitor might enter the first steps of the enzymatic reaction to promote inhibition. Using 2-C-vinyl-D-erythritol-2,4-cyclodiphosphate as only possible enzyme substrate, IspG is active in the first minutes of reaction in the presence of VEcPP, then the activity slowly decreases and after 15 minutes of reaction the enzyme is almost totally inhibited. Thus, VEcPP enters the active site and may undergo electron transfers before inhibiting the enzyme Escherichia coli

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.17.7.3 Fe2+ required Escherichia coli

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.17.7.3 2-C-methyl-D-erythritol 2,4-cyclodiphosphate + 2 reduced flavodoxin Escherichia coli
-
 (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate + H2O + 2 oxidized flavodoxin
-
 ?
1.17.7.3 2-C-methyl-D-erythritol 2,4-cyclodiphosphate + 2 reduced flavodoxin Escherichia coli K12
-
 (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate + H2O + 2 oxidized flavodoxin
-
 ?
1.17.7.3 additional information Escherichia coli IspG, as an interconnected enzymatic complex, promotes the conversion of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate in the presence of an external reduction system. In Escherichia coli, the natural flavodoxin (FldA)/flavodoxin reductase (FpR1)/NADPH system plays this role ?
-
 ?
1.17.7.3 additional information Escherichia coli K12 IspG, as an interconnected enzymatic complex, promotes the conversion of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate in the presence of an external reduction system. In Escherichia coli, the natural flavodoxin (FldA)/flavodoxin reductase (FpR1)/NADPH system plays this role ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.17.7.3 Escherichia coli P62620
-
-
1.17.7.3 Escherichia coli K12 P62620
-
-
1.17.7.3 no activity in Homo sapiens
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.17.7.3 recombinant His-tagged enzyme from Escherichia coli strain XL1-blue by nickel affinity chromatography, ultrafiltration, and gel filtration. Due to its oxygen sensitivity, Escherichia coli IspG needs to be handled in a glove box under a strictly inert (N2) atmosphere Escherichia coli

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.17.7.3 2-C-methyl-D-erythritol 2,4-cyclodiphosphate + 2 reduced flavodoxin
-
 Escherichia coli (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate + H2O + 2 oxidized flavodoxin
-
 ?
1.17.7.3 2-C-methyl-D-erythritol 2,4-cyclodiphosphate + 2 reduced flavodoxin
-
 Escherichia coli K12 (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate + H2O + 2 oxidized flavodoxin
-
 ?
1.17.7.3 additional information IspG, as an interconnected enzymatic complex, promotes the conversion of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate in the presence of an external reduction system. In Escherichia coli, the natural flavodoxin (FldA)/flavodoxin reductase (FpR1)/NADPH system plays this role Escherichia coli ?
-
 ?
1.17.7.3 additional information reaction under a strictly inert (N2) atmosphere. IspG, as an interconnected enzymatic complex, promotes the conversion of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate in the presence of an external reduction system, i.e. flavodoxin (FldA)/flavodoxin reductase (FpR1)/NADPH. The enzyme activity is evaluated by monitoring NADPH consumption at 340 nm in the presence of optimized concentrations of this reducing system Escherichia coli ?
-
 ?
1.17.7.3 additional information IspG, as an interconnected enzymatic complex, promotes the conversion of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate in the presence of an external reduction system. In Escherichia coli, the natural flavodoxin (FldA)/flavodoxin reductase (FpR1)/NADPH system plays this role Escherichia coli K12 ?
-
 ?
1.17.7.3 additional information reaction under a strictly inert (N2) atmosphere. IspG, as an interconnected enzymatic complex, promotes the conversion of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate in the presence of an external reduction system, i.e. flavodoxin (FldA)/flavodoxin reductase (FpR1)/NADPH. The enzyme activity is evaluated by monitoring NADPH consumption at 340 nm in the presence of optimized concentrations of this reducing system Escherichia coli K12 ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.17.7.3 GcpE
-
 Escherichia coli
1.17.7.3 IspG
-
 Escherichia coli

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.17.7.3 37
-
 assay at Escherichia coli

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.17.7.3 8
-
 assay at Escherichia coli

Cofactor

EC Number Cofactor Comment Organism Structure
1.17.7.3 flavodoxin
-
 Escherichia coli
1.17.7.3 [4Fe-4S] cluster Escherichia coli IspG harbors an oxygen-sensitive [4Fe-4S]2+ cluster in its catalytic site, that is held by three conserved cysteines and an additional non-cysteine ligand Escherichia coli

IC50 Value

EC Number IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
1.17.7.3 0.0627
-
 pH 8.0, 37°C Escherichia coli 2-C-vinyl-D-erythritol-2,4-cyclodiphosphate

General Information

EC Number General Information Comment Organism
1.17.7.3 metabolism enzyme IspG (GcpE) is an oxygen-sensitive [4Fe-4S] enzyme that catalyzes the penultimate step of the methylerythritol phosphate (MEP) pathway, overview. Whereas the mevalonate pathway occurs mostly in eukaryotes, the MEP pathway is used exclusively by important pathogens and is absent in humans Escherichia coli
1.17.7.3 additional information proposed mechanism for IspG, overview. The formal leaving of the cyclopyrophosphate ester at C-2 suggests possible formation of a carbocationic species as an initial intermediate. The carbocation could evolve towards the corresponding radical and carbanion by sequential electron transfers. A transient epoxide has also been proposed as primary intermediate, resulting from nucleophilic displacement of the leaving phosphate Escherichia coli
1.17.7.3 physiological function enzyme IspG (also called GcpE) is an oxygen-sensitive [4Fe-4S] enzyme catalyzing the penultimate step of the methylerythritol phosphate (MEP) pathway. It converts 2-C-methyl-d-erythritol-2,4-cyclo-diphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl-1-diphosphate (HMBPP). The reaction, assimilated to a reductive dehydration, involves redox partners responsible for the formal transfer of two electrons to substrate MEcPP. Proposed mechanism for IspG, overview Escherichia coli