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.

Biosynthesis of Ether-Type Polar Lipids in Archaea and Evolutionary Considerations

“…An early in vitro work on C 40 carotene synthesis in Halobacterium cutirubrum also showed that isoprenoids are synthesized from MVA via IPP, even though carotenes are not components of polar lipids (57). In vitro assays of enzyme activities of the pathway supported the conclusion led by the in vivo evidence.…”

brp and blh Are Required for Synthesis of the Retinal Cofactor of Bacteriorhodopsin in Halobacterium salinarum

“…This pathway was based on the in vitro reconstitution of ␤-carotene formation from mevalonate (32) and on the accumulation of C 40 -isoprenoid intermediates in colorless mutant strains that lacked ␤-carotene and retinal (33). However, direct biochemical or genetic evidence for the conversion of ␤-carotene to retinal has not been obtained.…”

Bacterioopsin-Mediated Regulation of Bacterioruberin Biosynthesis in Halobacterium salinarum

“…Early steps in the pathway are shared with the biosynthesis of phytanyl, the hydrophobic portion of the polar lipids that make up archaeal cell membranes, with the 20-carbon geranylgeranyl diphosphate (GGPP) being the last shared intermediate (7). In the retinal biosynthetic pathway, two GGPP molecules condense to form the 40-carbon carotenoid phytoene, which undergoes a series of desaturation reactions to form the red carotenoid lycopene (23). For retinal synthesis, lycopene cyclizes to form ␤-carotene, which is cleaved to form the 20-carbon retinal cofactor (23,42).…”

“…In the retinal biosynthetic pathway, two GGPP molecules condense to form the 40-carbon carotenoid phytoene, which undergoes a series of desaturation reactions to form the red carotenoid lycopene (23). For retinal synthesis, lycopene cyclizes to form ␤-carotene, which is cleaved to form the 20-carbon retinal cofactor (23,42). Alternatively, lycopene may be used as a precursor for bacterioruberins: tetrahydrobisanhydrobacterioruberin, bisanhydrobacterioruberin, and bacterioruberin (22,24).…”