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Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis

TitleNovel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis
Publication TypeArticolo su Rivista peer-reviewed
Year of Publication2014
AuthorsFrusciante, Sarah, Diretto Gianfranco, Bruno M., Ferrante Paola, Pietrella M., Prado-Cabrero A., Rubio-Moraga A., Beyer P., Gomez-Gomez L., Al-Babili S., and Giuliano Giovanni
JournalProceedings of the National Academy of Sciences of the United States of America
Keywordsaldehyde derivative, Amino Acid Sequence, article, beta cryptoxanthin, beta ionone, Biocatalysis, biosynthesis, carotenoid cleavage dioxygenase 1, carotenoid cleavage dioxygenase 2, Carotenoids, Catalysis, cellular distribution, chemical bond, chromoplast, controlled study, crocetin, crocetin dialdehyde, crocin, Crocus, Crocus sativus, cytoplasm, dioxygenase, Dioxygenases, endosperm, enzyme structure, Escherichia coli, in vitro study, Maize, Mass Spectrometry, Molecular Sequence Data, nonhuman, pistil stigma, prediction, priority journal, protein cleavage, protein expression, Substrate Specificity, Symmetric carotenoid cleavage, transcriptome, unclassified drug, Zeaxanthin, zeaxanthin cleavage dioxygenase, β-citraurin

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 C30 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.


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Citation KeyFrusciante201412246