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Literature summary for 1.17.7.3 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
Search for more literature for 1.17.7.3

Application

Application Comment Organism
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)

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

Inhibitors

Inhibitors Comment Organism Structure
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

Metals/Ions Comment Organism Structure
Fe2+ required Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
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
-
 ?
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
-
 ?
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 ?
-
 ?
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

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

Purification (Commentary)

Purification (Comment) Organism
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)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
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
-
 ?
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
-
 ?
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 ?
-
 ?
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 ?
-
 ?
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 ?
-
 ?
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

Synonyms Comment Organism
GcpE
-
 Escherichia coli
IspG
-
 Escherichia coli

Temperature Optimum [°C]

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

pH Optimum

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

Cofactor

Cofactor Comment Organism Structure
flavodoxin
-
 Escherichia coli
[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

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

General Information

General Information Comment Organism
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
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
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