Biosynthesis of isoprenoids: A bifunctional IspDF enzyme from Campylobacter jejuni

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Abstract

In the nonmevalonate pathway of isoprenoid biosynthesis, the conversion of 2C-methyl-D-erythritol 4-phosphate into its cyclic diphosphate proceeds via nucleotidyl intermediates and is catalyzed by the products of the ispD, ispE and ispF genes. An open reading frame of Campylobacter jejuni with similarity to the ispD and ispF genes of Escherichia coli was cloned into an expression vector directing the formation of a 42 kDa protein in a recombinant E. coli strain. The purified protein was shown to catalyze the transformation of 2C-methyl-D-erythritol 4-phosphate into 4-diphosphocytidyl-2C-methyl-D-erythritol and the conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate into 2C-methyl-D-erythritol 2,4-cyclodiphosphate at catalytic rates of 19 μmol·mg−1·min−1 and 7 μmol·mg−1·min−1, respectively. Both enzyme-catalyzed reactions require divalent metal ions. The C. jejuni enzyme does not catalyze the formation of 2C-methyl-D-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2C-methyl-D-erythritol, a side reaction catalyzed in vitro by the IspF proteins of E. coli and Plasmodium falciparum. Comparative genomic analysis show that all sequenced α- and η-proteobacteria have fused ispDF genes. These bifunctional proteins are potential drug targets in several human pathogens (e.g. Helicobacter pylori, C. jejuni and Treponema pallidum).

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