Characterization of a β-carotene isomerase from the cyanobacterium Cyanobacteria aponinum

Abstract

Carotenoids are essential components of the photosynthetic apparatus and precursors of plant hormones, such as strigolactones (SLs). SLs are involved in various aspects of plant development and stress-response processes, including the establishment of root and shoot architecture. SL biosynthesis begins with the reversible isomerization of all-trans-carotene into 9-cis-β-carotene, catalysed by DWARF27 β-carotene isomerase (D27). Sequence comparisons have revealed the presence of D27-related proteins in photosynthetic eukaryotes and cyanobacteria lacking SLs. To gain insight into the evolution of SL biosynthesis, we characterized the activity of a cyanobacterial D27 protein (CaD27) from Cyanobacterim aponinum, using carotenoid-accumulating Escherichia coli cells and in vitro enzymatic assays. Our results demonstrate that CaD27 is an all-trans/cis and cis/cis-β-carotene isomerase, with a cis/cis conversion preference. CaD27 catalysed 13-cis/15-cis-, all-trans/9-cis-β-carotene, and neurosporene isomerization. Compared with plant enzymes, it exhibited a lower 9-cis-/all-trans-β-carotene conversion ratio. A comprehensive genome survey revealed the presence of D27 as a single-copy gene in the genomes of 20 out of 200 cyanobacteria species analysed. Phylogenetic and enzymatic analysis of CaD27 indicated that cyanobacterial D27 genes form a single orthologous group, which is considered an ancestral type of those found in photosynthetic eukaryotes. This article is part of the theme issue 'The evolution of plant meta‌bolism'.

Keywords: DWARF27 β-carotene isomerase; carotenoids; cyanobacteria; strigolactones.

Figure 1.

Ultra-high performance liquid chromatography (UHPLC) analysis of in vitro assays performed with crude lysates of BL21(DE3) Escherichia coli cells expressing thioredoxin-CaD27 (CaD27), thioredoxin-AtD27 (Arabidopsis thaliana D27), thioredoxin-AtD27Like1 (AtD27-Like1) or thioredoxin (Control) with different β-carotene isomers. Left: (a) chromatograms of the incubations with different β-carotene isomers. Right: the relative peak surface of the different β-carotene isomers separated in the chromatograms; (b) all-trans-β-carotene (peak I); (c) 9-cis-β-carotene (peak II); (d) 13-cis-β-carotene (peak III) and (e) 15-cis-β-carotene (peak IV). The sum of all β-carotene peaks is considered as 100%. UV–visible spectra are depicted in the insets. An ANOVA was performed to determine significance (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars represent the s.d.

Figure 2.

Non-root neighbour-joining tree of D27 orthologues in Archaeplastida. Subtrees designated with black triangles are depicted in electronic supplementary material, figure S4. Information on sequences used for this study is given in electronic supplementary material, table S3. Bootstrap values of more than 50% are shown.