title: Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis.
abstract: 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.
authors.affiliation: Italian National Agency for New Technologies, Energy, and Sustainable Development, Casaccia Research Centre, 00123 Rome, Italy;Sapienza, University of Rome, 00185 Rome, Italy;
authors.forename: Sarah
authors.initials: S
authors.lastname: Frusciante
authors.affiliation: Italian National Agency for New Technologies, Energy, and Sustainable Development, Casaccia Research Centre, 00123 Rome, Italy;
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Diretto
authors.affiliation: Faculty of Biology, University of Freiburg, D-79104 Freiburg, Germany;
authors.forename: Mark
authors.initials: M
authors.lastname: Bruno
authors.affiliation: Italian National Agency for New Technologies, Energy, and Sustainable Development, Casaccia Research Centre, 00123 Rome, Italy;
authors.forename: Paola
authors.initials: P
authors.lastname: Ferrante
authors.affiliation: Italian National Agency for New Technologies, Energy, and Sustainable Development, Casaccia Research Centre, 00123 Rome, Italy;
authors.forename: Marco
authors.initials: M
authors.lastname: Pietrella
authors.affiliation: Center for Desert Agriculture, Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia; and.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Prado-Cabrero
authors.affiliation: Instituto Botánico, Facultad de Farmacia, Universidad de Castilla-La Mancha, 02071 Albacete, Spain.
authors.forename: Angela
authors.initials: A
authors.lastname: Rubio-Moraga
authors.affiliation: Faculty of Biology, University of Freiburg, D-79104 Freiburg, Germany;
authors.forename: Peter
authors.initials: P
authors.lastname: Beyer
authors.affiliation: Instituto Botánico, Facultad de Farmacia, Universidad de Castilla-La Mancha, 02071 Albacete, Spain.
authors.forename: Lourdes
authors.initials: L
authors.lastname: Gomez-Gomez
authors.affiliation: Faculty of Biology, University of Freiburg, D-79104 Freiburg, Germany;Center for Desert Agriculture, Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia; and.
authors.forename: Salim
authors.initials: S
authors.lastname: Al-Babili
authors.affiliation: Italian National Agency for New Technologies, Energy, and Sustainable Development, Casaccia Research Centre, 00123 Rome, Italy; giovanni.giuliano@enea.it.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Giuliano
mesh_terms: D055162:Biocatalysis; D002338:Carotenoids / Q000096:biosynthesis*; D027622:Crocus / Q000201:enzymology / Q000378:metabolism*; D049308:Dioxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D013379:Substrate Specificity
chemical_list: D002338:Carotenoids; C029036:crocin; D049308:Dioxygenases; C431939:carotenoid cleavage dioxygenase 1
keywords: symmetric carotenoid cleavage; β-citraurin
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