Isopentenyl diphosphate biosynthesis via a mevalonate-independent pathway:Isopentenyl monophosphate kinase catalyzes the terminal enzymatic step

In plants, the biosynthesis of isopentenyl diphosphate, the central precurs or of all isoprenoids, proceeds via two separate pathways. The cytosolic co mpartment harbors the mevalonate pathway, whereas the newly discovered deox yxylulose 5-phosphate pathway, which also operates in certain eubacteria, i ncluding Escherichia coli, is localized to plastids. Only the first two ste ps of the plastidial pathway, which involve the condensation of pyruvate an d glyceraldehyde 3-phosphate to deoxyxylulose 5-phosphate followed by intra molecular rearrangement and reduction to 2-C-methylerythritol 4-phosphate, have been established. Here we report the cloning from peppermint(Mentha x piperita) and E. coli, and expression, of a kinase that catalyzes the phosp horylation of isopentenyl monophosphate as the last step of this biosynthet ic sequence to isopentenyl diphosphate. The plant gene defines an ORF of 1, 218 bp that, when the proposed plastidial targeting sequence is excluded, c orresponds to approximate to 308 aa with a mature size of approximate to 33 kDa. The E. coli gene (ychB), which is located at 27.2 min of the chromoso mal map, consists of 852 nt, encoding a deduced enzyme of 283 aa with a siz e of 31 kDa. These enzymes represent a conserved class of the GHMP family o f kinases, which includes galactokinase. homoserine kinase, mevalonate kina se, and phosphomevalonate kinase. with homologues in plants and several eub acteria. Besides the preferred substrate isopentenyl monophosphate, the rec ombinant peppermint and E. coli kinases also phosphorylate isopentenol, and , much less efficiently, dimethylallyl alcohol, but dimethylallyl monophosp hate does not serve as a substrate. Incubation of secretory cells isolated from peppermint glandular trichomes with isopentenyl monophosphate resulted in the rapid production of monoterpenes and sesquiterpenes, confirming tha t isopentenyl monophosphate is the physiologically relevant, terminal inter mediate of the deoxyxylulose 5-phosphate pathway.