Analysis in vitro of the enzyme CRTISO establishes a poly-cis-carotenoid biosynthesis pathway in plants

Abstract

Most enzymes in the central pathway of carotenoid biosynthesis in plants have been identified and studied at the molecular level. However, the specificity and role of cis-trans-isomerization of carotenoids, which occurs in vivo during carotene biosynthesis, remained unresolved. We have previously cloned from tomato (Solanum lycopersicum) the CrtISO gene, which encodes a carotene cis-trans-isomerase. To study the biochemical properties of the enzyme, we developed an enzymatic in vitro assay in which a purified tomato CRTISO polypeptide overexpressed in Escherichia coli cells is active in the presence of an E. coli lysate that includes membranes. We show that CRTISO is an authentic carotene isomerase. Its catalytic activity of cis-to-trans isomerization requires redox-active components, suggesting that isomerization is achieved by a reversible redox reaction acting at specific double bonds. Our data demonstrate that CRTISO isomerizes adjacent cis-double bonds at C7 and C9 pairwise into the trans-configuration, but is incapable of isomerizing single cis-double bonds at C9 and C9'. We conclude that CRTISO functions in the carotenoid biosynthesis pathway in parallel with zeta-carotene desaturation, by converting 7,9,9'-tri-cis-neurosporene to 9'-cis-neurosporene and 7'9'-di-cis-lycopene into all-trans-lycopene. These results establish that in plants carotene desaturation to lycopene proceeds via cis-carotene intermediates.

Figure 1.

HPLC analysis of carotenes extracted from E. coli cells accumulating cis-ζ-carotene in the absence (A) and presence (B) of CRTISO. To identify the isomers, the sample in B was mixed with a synthetic all-trans-ζ-carotene and analyzed by HPLC (C). MaxPlot chromatograms showing each peak at its λ_max are presented. Peak 1, All-trans-ζ-carotene; peak 2, 9,15,9′-tri-cis-ζ-carotene; peak 3, 9,9′-di-cis-ζ-carotene; peaks 4a and 4b are cis-isomers of phytofluene; peak 5, 15-cis-phytoene. Absorption spectra of specific peaks are presented to the boxes. 9,15,9′-Tri-cis-ζ-carotene is distinguished from the 9,9′-cis-isomer by the typical absorbance at 297.5 nm.

Figure 2.

Expression of CRTISO in E. coli. Proteins were separated by SDS-PAGE and stained with Coomassie blue. Lane 1, Insoluble fraction (pellet) of a lysate from E. coli carrying pQE60; lane 2, soluble fraction (supernatant) of E. coli carrying pQE60; lane 3, insoluble fraction of E. coli carrying pCrtISO654; lane 4, soluble fraction of E. coli carrying pCrtISO654; lane 5, column-purified CRTISO polypeptide.

Figure 3.

HPLC analysis of cis-carotene substrates and products in a CRTISO in vitro assay. A, Carotenes at the beginning of the reaction (time 0 min); B, after 90 min. Peak 1a, trans-Lycopene; peak 1b, di-cis-lycopene; peak 2, prolycopene; peak 3a, neurosporene, isomer 1; peak 3b, neurosporene, isomer 2; peak 3c, 7,9,9′-cis-isomer of neurosporene; peak 4a, 9,15,9′-cis-ζ-carotene; peak 4b, 9,9′-cis-ζ-carotene isomer; peak 5a and 5b are β-carotene isomers; peak 6, phytofluene; peak 7, phytoene.

Figure 4.

A, Time course of prolycopene isomerization in vitro. A mixture of cis-carotenes from tangerine, at a final concentration of 180 μg mL⁻¹, was used as a substrate in the assay with lysate of E. coli that expressed CRTISO. Aliquots were subjected to HPLC analysis at the indicated time points. Dashed line, Prolycopene; dotted line, 7,9-di-cis-lycopene; solid line, trans-lycopene. B, Reaction carried out with purified 7,9,9′-tri-cis-neurosporene as a substrate. Solid line, 7,9, 9′-Tri-cis-neurosporene; dashed line, neurosporene isomer 1; dotted line, neurosporene isomer 2; dotted-dashed line, trans-neurosporene.

Figure 5.

Effect of redox components on the activity in vitro of CRTISO. Time-course experiments were run and plotted as in Figure 4A in the presence of the different effectors. For each condition, the area under the curves was calculated. CRTISO activity in the control condition was taken as 1.0. A, Requirement for respiratory redox intermediates; black bars represent nondialyzed E. coli lysate (supernatant) and gray bars, dialyzed preparations. B, Influence of anaerobic conditions and ferricyanide. C, Effect of various oxidized quinones on CRTISO activity was measured in the absence (black bars) or presence (gray bars) of 1 mm NADH. BQ, benzoquinone; DQ, duroquinone; UQ, ubiquinone; PQ, plastoquinone (1 mm each).

Figure 6.

Proposed pathway of carotenoid biosynthesis in plants. CRTISO, Carotene isomerase; CRTR-B, β-ring hydroxylase, CRTR-E, ɛ-ring hydroxylase; GGPP, geranylgeranyl diphosphate; LCY-B, lycopene β-cyclase; LCY-E, lycopene ɛ-cyclase; PDS, phytoene desaturase; PSY, phytoene synthase; ZDS, ζ-carotene desaturase; ZEP, zeaxanthin epoxidase.