Crocus sativus stigmas are the source of the saffron spice and accumulate the apocarotenoids crocetin, crocins, picrocrocin, and safranal, responsible for its color, taste, and aroma. Through deep transcriptome sequencing, we identified a novel dioxygenase, carotenoid cleavage dioxygenase 2 (CCD2), expressed early during stigma development and closely related to, but distinct from, the CCD1 dioxygenase family. CCD2 is the only identified member of a novel CCD clade, presents the structural features of a bona fide CCD, and is able to cleave zeaxanthin, the presumed precursor of saffron apocarotenoids, both in Escherichia coli and in maize endosperm. The cleavage products, identified through high-resolution mass spectrometry and comigration with authentic standards, are crocetin dialdehyde and crocetin, respectively. In vitro assays show that CCD2 cleaves sequentially the 7,8 and 7′,8′ double bonds adjacent to a 3-OH-β-ionone ring and that the conversion of zeaxanthin to crocetin dialdehyde proceeds via the C 30 intermediate 3-OH-β-apo-8′-carotenal. In contrast, zeaxanthin cleavage dioxygenase (ZCD), an enzyme previously claimed to mediate crocetin formation, did not cleave zeaxanthin or 3-OH-β-apo-8′-carotenal in the test systems used. Sequence comparison and structure prediction suggest that ZCD is an N-truncated CCD4 form, lacking one blade of the β-propeller structure conserved in all CCDs. These results constitute strong evidence that CCD2 catalyzes the first dedicated step in crocin biosynthesis. Similar to CCD1, CCD2 has a cytoplasmic localization, suggesting that it may cleave carotenoids localized in the chromoplast outer envelope.β-citraurin | symmetric carotenoid cleavage

Opsins are membrane photoreceptors closely related to the heat-shock proteins of the HSP30 family. Their functions include light-driven ion pumping in archaea and light detection in algae and animals, using the apocarotenoid retinal as a light-absorbing prosthetic group. We describe a gene of Fusarium fujikuroi, carO, coding for a polypeptide resembling opsins and HSP30-like proteins and contiguous to the genes of the carotenoid pathway, carRA and carB. Transcription of carO is induced by light and is deregulated in carotenoid-overproducing mutants. The same regulation pattern is exhibited by carRA and carB; and common conserved DNA elements are found in the three promoters. Heat shock resulted in a modest induction of carO transcription, similar to the one exhibited by carB, confirming a common regulation. Targeted mutagenesis of carO produced no apparent phenotypic modification, including no change in the photoinduction of carotenoid biosynthesis.

The car gene cluster of the ascomycete Fusarium fujikuroi encodes two enzymes responsible for torulene biosynthesis (CarRA and CarB), an opsin-like protein (CarO), and a putative carotenoid cleaving enzyme (CarX). It was presumed that CarX catalyzes the formation of the major carotenoid in F. fujikuroi, neurosporaxanthin, a cleavage product of torulene. However, targeted deletion of carX did not impede neurosporaxanthin biosynthesis. On the contrary, ⌬carX mutants showed a significant increase in the total carotenoid content, indicating an involvement of CarX in the regulation of the pathway. In this work, we investigated the enzymatic activity of CarX. The expression of the enzyme in ␤-carotene-accumulating Escherichia coli cells led to the formation of the opsin chromophore retinal. The identity of the product was proven by high-performance liquid chromatography and gas chromatography-mass spectrometry. Subsequent in vitro assays with heterologously expressed and purified CarX confirmed its ␤-carotene-cleaving activity and revealed its capability to produce retinal also from other substrates, such as ␥-carotene, torulene, and ␤-apo-8-carotenal. Our data indicate that the occurrence of at least one ␤-ionone ring in the substrate is required for the cleavage reaction and that the cleavage site is determined by the distance to the ␤-ionone ring. CarX represents the first retinal-synthesizing enzyme reported in the fungal kingdom so far. It seems likely that the formed retinal is involved in the regulation of the carotenoid biosynthetic pathway via a negative feedback mechanism.Carotenoids are widespread lipophilic pigments synthesized by all photosynthetic organisms and some nonphotosynthetic fungi and bacteria. These yellow, orange, and red isoprenoid compounds fulfill diverse functions in all taxa. In addition, carotenoids are the precursors of several physiologically essential compounds, like the ubiquitous chromophore retinal, the phytohormone abscisic acid (ABA), and the fungal sex hormone trisporic acid.The synthesis of these carotenoid cleavage products, known as apocarotenoids, is catalyzed in general by carotenoid oxygenases, which constitute a new nonheme iron enzyme family common in all taxa (for reviews, see references 1, 11, 15, and 24). Recently, the crystal structure of a member of this family, the Synechocystis apocarotenoid oxygenase, was elucidated at 2.4-Å resolution. The enzyme contains an Fe 2ϩ -4-His arrangement at the axis of a seven-bladed ␤-propeller chain fold covered by a dome formed by six large loops (20). It has also been shown that the 15,15Ј BCO I (␤-carotene oxygenase I) acts as a monooxygenase (21). However, recent investigations of AtCCD1 (Arabidopsis thaliana carotenoid cleavage dioxygenase 1) suggested a dioxygenase mechanism (31).VP14 (Viviparous 14), a maize enzyme mediating the oxidative cleavage of 9-cis-violaxanthin and 9Ј-cis-neoxanthin to form the precursor of ABA, xanthoxin, was the first molecularly identified carotenoid oxygenase (34). Data from sequenced genomes hav...

The presence of hypoxic regions in solid tumors is an adverse prognostic factor for patient outcome. Here, we show that hypoxia induces the expression of Ephrin-A3 through a novel hypoxia-inducible factor (HIF)-mediated mechanism. In response to hypoxia, the coding EFNA3 mRNA levels remained relatively stable, but HIFs drove the expression of previously unknown long noncoding (Inc) RNAs from EFNA3 locus and these IncRNA caused Ephrin-A3 protein accumulation. Ephrins are cell surface proteins that regulate diverse biological processes by modulating cellular adhesion and repulsion. Mounting evidence implicates deregulated ephrin function in multiple aspects of tumor biology. We demonstrate that sustained expression of both Ephrin-A3 and novel EFNA3 lncRNAs increased the metastatic potential of human breast cancer cells, possibly by increasing the ability of tumor cells to extravasate from the blood vessels into surrounding tissue. In agreement, we found a strong correlation between high EFNA3 expression and shorter metastasis-free survival in breast cancer patients. Taken together, our results suggest that hypoxia could contribute to metastatic spread of breast cancer via HIF-mediated induction of EFNA3 lncRNAs and subsequent Ephrin-A3 protein accumulation.