Enzymatic synthesis of C40 carotenes by cell-free preparation from Halobacterium cutirubrum

Abstract

[14C]Mevalonate or (14C)isopentenyl pyrophosphate was found to be converted to transphytoene, trans-phytofluene, lycopene, and beta-carotene by a cell-free 270 000 X g supernatant fraction prepared from Halobacterium cutirubrum cells that were broken by manual grinding with glass beads. Incubations were done under N2 in the dark at 37 degrees C in 4 M NaCl in presence of FAD, NADP, and MgCl2; ATP was also added when mevalonate was the substrate. This system was also capable of converting trans-(14C)phytoene to beta-carotene via the intermediates trans-phytofluene, zeta-carotene, neurosporene, lycopene, and gamma-carotene. Each of these labelled intermediates on incubation separately with the same enzyme system was shown to be converted to the intermediates farther down the pathway. The results of this study show that the biosynthetic pathway for the formation of C40 carotenes in H. cutirubrum proceeds as follows: isopentenyl pyrophosphate leads to trans-phytoene leads to trans-phytofluene leads to zeta-carotene leads to neurosporene leads to lycopene leads to gamma-carotene leads to beta-carotene. This pathway differs from that in higher plants in that the cis isomers of phytoene and phytofluene are not on the main pathway of carotene biosynthesis, as they are in higher plants. Furthermore, trans-phytoene, which has not been reported to have any role in higher plants, appears to be the main intermediate in carotene biosynthesis in H. cutirubrum.