Studies on the nonmevalonate pathway of terpene biosynthesis - The role of2C-methyl-D-erythritol 2,4-cyclodiphosphate in plants

2C-Methyl-D-erythritol 2,4-cyclodiphosphate was recently shown to be formed from 2C-methyl-D-erythritol 4-phosphate by the consecutive action of IspD, IspE, and IspF proteins in the nonmevalonate pathway of terpenoid biosynth esis. To complement previous work with radiolabelled precursors, we have no w demonstrated that [U-C-13(5)]2C-methyl-D-erythritol 4-phosphate affords [ U-C-13(5)]2C-methyl-D-erythritol 2,4-cyclodiphosphate in isolated chromopla sts of Capsicum annuum and Narcissus pseudonarcissus. Moreover, chromoplast s are shown to efficiently convert 2C-methyl-D-erythritol 4-phosphate as we ll as 2C-methyl-D-erythritol 2,4-cyclodiphosphate into the carotene precurs or phytoene. The bulk of the kinetic data collected in competition experime nts with radiolabeled substrates is consistent with the notion that the cyc lodiphosphate is an obligatory intermediate in the nonmevalonate pathway to terpenes. Studies with [2,2'-C-13(2)]2C-methyl-D-erythritol 2,4-cyclodipho sphate afforded phytoene characterized by pairs of jointly transferred C-13 atoms in the positions 17/1, 18/5, 19/9, and 20/13 and, at a lower abundan ce, in positions 16/1, 4/5, 8/9, and 12/13. A detailed scheme is presented for correlating the observed partial scrambling of label with the known lac k of fidelity of the isopentenyl diphosphate/dimethylethyl diphosphate isom erase.