Sll0254 (CrtL(diox)) is a bifunctional lycopene cyclase/dioxygenase in cyanobacteria producing myxoxanthophyll - PubMed (original) (raw)

Sll0254 (CrtL(diox)) is a bifunctional lycopene cyclase/dioxygenase in cyanobacteria producing myxoxanthophyll

Hatem E Mohamed et al. J Bacteriol. 2006 May.

Abstract

Upon depletion of Sll0254 in Synechocystis sp. strain PCC 6803, cyclized carotenoids were replaced by linear, relatively hydrophilic carotenoids, and the amount of the two photosystems decreased greatly. Full segregants of the sll0254 deletion in Synechocystis were not obtained, implying that this gene is essential for survival, most likely to allow normal cell division. The N-terminal half of Sll0254 has limited similarity to the family of lycopene cyclases, has an additional dehydrogenase motif near the N terminus, and is followed by a Rieske 2Fe-2S center sequence signature. To test whether Sll0254 serves as a lycopene cyclase in Synechocystis, the corresponding gene was expressed in Escherichia coli strains that can produce lycopene or neurosporene. In the presence of Sll0254 these linear carotenoids were converted into cyclized, relatively hydrophilic pigments, with masses consistent with the introduction of two hydroxyl groups and with spectra indicative of only small changes in the number of conjugated double bonds. This suggests that Sll0254 catalyzes formation of oxygenated, cyclized carotenoids. We interpret the appearance of the hydroxyl groups in the carotenoids to be due to dioxygenase activity involving the Rieske 2Fe-2S center and the additional dehydrogenase domain. This dioxygenase activity is required in the myxoxanthophyll biosynthesis pathway, after or concomitant with cyclization on the other end of the molecule. We interpret Sll0254 to be a dual-function enzyme with both lycopene cyclase and dioxygenase activity and have named it CrtL(diox).

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Figures

FIG. 1.

FIG. 1.

The 77 K fluorescence emission spectra of the wild type (solid bold line) and the Δsll0254Sp transformant early after transformation (broken line) and after several more subcultures (solid thin line) in the presence of increasing concentrations of spectinomycin. Cells were grown at low-light intensity (0.5 μmol of photons m−2 s−1). Each sample contained the same cell concentration. Fluorescence excitation was at 435 nm.

FIG. 2.

FIG. 2.

HPLC fractionation of methanol/ammonia extracts from the Δsll0254Sp transformant grown in medium supplemented with 50 (A) or 200 (B) μg/ml spectinomycin and from wild-type Synechocystis sp. strain PCC 6803 (C). All cultures were grown in continuous light at a light intensity of 0.5 μmol of photons m−2 s−1. The main pigments from the wild-type strain have been identified: M, myxoxanthophyll; Z, zeaxanthin; E, echinenone; β, β-carotene; and Ch, chlorophyll a. The absorption spectra of the pigments accumulated in peaks 1 to 3 are shown in Fig. 3.

FIG. 3.

FIG. 3.

Absorption spectra of pigments found in the Δsll0254Sp transformant. Spectrum numbers refer to pigment peaks identified in Fig. 2.

FIG.4.

FIG.4.

HPLC chromatograms of methanol extracts from E. coli strain DH5α harboring pAC-NEUR (A) or pAC-LYC (B) and carrying pET16b-sll0254 (top) or the empty pET16b plasmid vector (bottom). N, neurosporene; L, lycopene. The absorption spectra of the pigments accumulated in the numbered peaks are shown in Fig. 5.

FIG. 5.

FIG. 5.

Absorption spectra of carotenoids present in HPLC peaks 1, 2, and 3 (Fig. 4) that appear upon pET16b-sll0254 introduction in E. coli carrying pAC-NEUR (A) or pAC-LYC (B).

FIG. 6.

FIG. 6.

Transmission electron micrographs of cells of wild-type Synechocystis sp. strain PCC 6803 (A) and the Δsll0254Sp transformant (B and C). The cell division plate (black arrow) and secondary cell division plates (white arrow) have been indicated in cells of the transformant. Scale bar, 1 μm.

FIG. 7.

FIG. 7.

Alignment of sections of Sll0254 with domains of members of the lycopene cyclase family and of γ-carotene desaturase (CrtU) from C. tepidum. (A) Shown are two desaturase motifs in the N-terminal region; the first motif is present in Sll0254 and in CrtU from C. tepidum, whereas the other is conserved in members of the lycopene cyclase family as well. (B) The TGY motif that is conserved in members of the lycopene cyclase family and, with a conserved modification, in Sll0254 but not in CrtU. (C) A region toward the C-terminal end of one of the lycopene cyclase family members, neoxanthin synthase from potato, that is reasonably conserved in a domain near the middle of Sll0254 but that is not found convincingly in C. tepidum CrtU. Asterisks indicate residues that are conserved between Sll0254 and four (A and B) or one (C) member of the lycopene cyclase family, colons signify conservation of residues between Sll0254 and more than one member of the lycopene cyclase family shown here, periods indicate conservation between Sll0254 and one member of the lycopene cyclase family shown here, and vertical lines indicate identity between Sll0254 and C. tepidum CrtU. Alignments were made using the DIALIGN program that is suitable for determining local similarities (20). St NS, neoxanthin synthase from Solanum tuberosum (potato); Te EC, ɛ-cyclase from Tagetes erecta (marigold); Sg LC, lycopene cyclase from Streptomyces griseus; Pa LC, lycopene cyclase (CrtY) from Pantoea agglomerans; and Ct CD, γ-carotene desaturase (CrtU) from C. tepidum.

FIG. 8.

FIG. 8.

Schematic representation of lycopene conversion to deoxymyxol by CrtD and Sll0254 in Synechocystis sp. strain PCC 6803 (left) and to 1′,2′-dihydroxy-γ-carotene by Sll0254 in an E. coli strain that produces lycopene (right).

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