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Preparation of Quality Inositol Pyrophosphates
10:34

Preparation of Quality Inositol Pyrophosphates

Published on: September 3, 2011

2C-Methyl-d-erythritol 4-phosphate enhances and sustains cyclodiphosphate synthase IspF activity

J Kipchirchir Bitok1, Caren Freel Meyers

  • 1Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.

ACS Chemical Biology
|July 31, 2012
PubMed

Insights

Isoprenoid biosynthesis in pathogens is regulated by a novel mechanism. The enzyme cyclodiphosphate synthase (IspF) is activated by MEP and inhibited by FDP, suggesting new therapeutic targets.

Area of Science:

  • Biochemistry
  • Enzymology
  • Metabolic Pathways

Background:

  • Understanding isoprenoid biosynthesis regulation in human pathogens is crucial for developing new antimicrobial strategies.
  • The Methylerythritol Phosphate (MEP) pathway is essential for isoprenoid production in many pathogens.
  • Limited knowledge exists regarding the regulatory mechanisms governing the MEP pathway.

Purpose of the Study:

  • To investigate the hypothesis that isoprenoid biosynthesis is regulated by feedback inhibition of cyclodiphosphate synthase (IspF) by downstream metabolites.
  • To elucidate the role of 2C-methyl-d-erythritol 4-phosphate (MEP) and downstream isoprenoid diphosphates in regulating IspF activity.

Main Methods:

  • Recombinant E. coli IspF enzyme was purified and characterized.
  • Enzyme activity assays were performed using various concentrations of MEP and downstream metabolites (IDP, DMADP, GDP, FDP).
  • The effect of MEP and FDP on IspF activity and complex formation was analyzed.

Main Results:

  • Recombinant IspF was not inhibited by downstream metabolites (IDP, DMADP, GDP, FDP) under standard conditions.
  • MEP, the first committed intermediate of the MEP pathway, activated and sustained IspF activity.
  • The IspF-MEP complex was found to be inhibited by FDP, indicating a feedback inhibition mechanism.
  • The methylerythritol scaffold unique to this pathway was identified as the driver for IspF activation and stabilization.

Conclusions:

  • A novel feed-forward regulatory mechanism for MEcDP production exists, involving MEP activation of IspF.
  • Isoprenoid biosynthesis is regulated via feedback inhibition of the IspF-MEP complex by FDP.
  • The IspF-MEP complex represents a potential therapeutic target for developing inhibitors against essential isoprenoid biosynthesis in pathogens.

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