Synthesis of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate and kinetic studies of Mycobacterium tuberculosis IspF

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.

Figure 1

MEP biosynthetic pathway.

Figure 2

Enantiomeric synthesis of 6.

Figure 3

Partial alignment, purification and characterization of IspF. Panel A: Partial alignment of putative M. tuberculosis (MTB) IspF and E. coli (ECO) IspF. Identities are indicated in black boxes and similarities in gray. Conserved amino acids reported to be involved in substrate specificity (#) and the Zn²⁺ binding (*) are indicated. Panel B: Expression and purification of His tagged M. tuberculosis IspF. SDS-PAGE and Western blot analysis of protein fractions from E. coli transfomed with pET28a(+)::Rv3581c. Lane 1, cell lysate prior to IPTG induction. Lane 2, cell lysate after IPTG treatment. Lane 3, purified His-tagged IspF visualized by Coomassie Brilliant Blue 250R. Lane 4, Western blot analysis of purified IspF using an anti-His antibody. Panel C: The effect of CDP-ME2P concentration on M. tuberculosis IspF activity. Reaction mixtures are described in the Experimental Procedures. ADP generated from this reaction was detected using ADP Quest HS Kinase kit and a Synergy^™ HT Multi-Detection Microplate Reader with an excitation wavelength of 520 nm and emission wavelength of 590 nm.

Figure 4

Determination of activity of recombinant M. tuberculosis IspF. Three different reaction mixtures containing the indicated compounds were analyzed. The arrows indicate the migration of authentic compounds.