Synthesis of 4-Diphosphocytidyl-2-C-Methyl-D-Erythritol 2-Phosphate and Kinetic Studies of Mycobacterium tuberculosis IspF

Prabagaran Narayanasamy, Hyungjin Eoh, Patrick J. Brennan, Dean C. Crick

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Many pathogenic bacteria utilize the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate, two major building blocks of isoprenoid compounds. The fifth enzyme in the MEP pathway, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (ME-CPP) synthase (IspF), catalyzes the conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P) to ME-CPP with a corresponding release of cytidine 5-monophosphate (CMP). Because there is no ortholog of IspF in human cells, IspF is of interest as a potential drug target. However, study of IspF has been hindered by a lack of enantiopure CDP-ME2P. Herein, we report the first, to our knowledge, synthesis of enantiomerically pure CDP-ME2P from commercially available D-arabinose. Cloned, expressed, and purified M. tuberculosis IspF was able to utilize the synthetic CDP-ME2P as a substrate, a result confirmed by mass spectrometry. A convenient, sensitive, in vitro IspF assay was developed by coupling the CMP released during production of ME-CPP to mononucleotide kinase, which can be used for high throughput screening.

Original languageEnglish (US)
Pages (from-to)117-122
Number of pages6
JournalChemistry and Biology
Volume17
Issue number2
DOIs
StatePublished - Feb 26 2010

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Mycobacterium tuberculosis
Cytidine Monophosphate
Cytidine
Kinetics
Arabinose
Biosynthesis
Terpenes
Mass spectrometry
Assays
Mass Spectrometry
Bacteria
Screening
Phosphotransferases
Cells
Throughput
Substrates
Enzymes
Pharmaceutical Preparations
4-diphosphocytidyl-2C methylerythritol 2-phosphate
methyl-D-erythritol 2,4-cyclodiphosphate

Keywords

  • CHEMBIO
  • MCIROBIO

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

Cite this

Synthesis of 4-Diphosphocytidyl-2-C-Methyl-D-Erythritol 2-Phosphate and Kinetic Studies of Mycobacterium tuberculosis IspF. / Narayanasamy, Prabagaran; Eoh, Hyungjin; Brennan, Patrick J.; Crick, Dean C.

In: Chemistry and Biology, Vol. 17, No. 2, 26.02.2010, p. 117-122.

Research output: Contribution to journalArticle

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