The identification of isoprenoids that bind in the intersubunit cavity of Escherichia coli 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase by complementary biophysical methods

The discovery of a distinct metabolic pathway, the non-mevalonate or 1-deoxy-D-xylulose-5-phosphate ( DOXP) pathway for isoprenoid precursor biosynthesis, in eubacteria and apicomplexan parasites has revealed a new set of potential drug targets. The emphasis of research on this pathway has been on delineating the intermediates and the biochemical and structural characterization of component enzymes. Two new monoclinic crystal forms of recombinant Escherichia coli 2C-methyl-D-erythritol-2,4-cyclodiphosphate ( MECP) synthase cocrystallized with (i) CMP and (ii) CMP and MECP show well defined electron density at the subunit interface suggestive of an isoprenoid-like ligand. P-31 NMR analysis of the recombinant protein sample indicates the presence of bound diphosphate species and electrospray mass spectrometry identifies a mixture of isopentenyl diphosphate (and/or dimethylallyl diphosphate), geranyl diphosphate and farnesyl diphosphate in an approximate ratio of 1: 4: 2. The most prevalent species, geranyl diphosphate, was successfully modelled into the electron density, revealing the important protein - ligand interactions that stabilize binding of the isoprenoid. The observation that MECP synthase binds three metabolites that are produced by enzymes two, three and four stages downstream in isoprenoid biosynthesis suggests that feedback regulation of the non-mevalonate pathway is possible.