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

Handle URI:
http://hdl.handle.net/10754/563692
Title:
Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis
Authors:
Frusciante, Sarah; Diretto, Gianfranco; Bruno, Mark; Ferrante, Paola; Pietrella, Marco; Prado-Cabrero, Alfonso; Rubio-Moraga, Ángela L.; Beyer, Peter D.; Gómez-Gómez, Lourdes; Al-Babili, Salim ( 0000-0003-4823-2882 ) ; Giuliano, Giovanni
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.
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division; Center for Desert Agriculture; Environmental Science and Engineering Program; Bioscience Program
Publisher:
Proceedings of the National Academy of Sciences
Journal:
Proceedings of the National Academy of Sciences
Issue Date:
5-Aug-2014
DOI:
10.1073/pnas.1404629111
PubMed ID:
25097262
PubMed Central ID:
PMC4143034
Type:
Article
ISSN:
00278424
Sponsors:
We thank Hansgeorg Ernst for providing the synthetic substrates; Chiara Lico for a gift of N. benthamiana plants and for helpin agroinfiltration experiments; Elena Romano and Emanuela Viaggiu at the Centre of Advanced Microscopy "Patrizia Albertano" for the confocal images; Gaetano Perrotta, Paolo Facella, and Fabrizio Carbone for 454 sequencing; and Alessia Fiore for critical reading of the manuscript. This work was supported by the Italian Ministry of Research (Project "Integrated Knowledge for the Sustainability and Innovation of Italian Agri-Food"), German Research Foundation (Deutsche Forschungsgemeinschaft) Grant AL 892/1-4, the European Union [The development of tools and effective strategies for the optimisation of useful secondary metabolite production in planta, Seventh Framework Programme (FP7) Contract 244348; From discovery to products: A next generation pipeline for the sustainable generation of high-value plant products, FP7 Contract 613153], the King Abdullah University of Science and Technology, the Spanish Ministerio de Ciencia e Innovacion (BIO2009-07803), and the Iberoamerican Network for the Study of Carotenoids as Food Ingredients (112RT0445). S. F. was supported by short-term fellowships of the PlantEngine (FA1006) and Saffronomics (FA1101) European Cooperation in Science and Technology actions.
Additional Links:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4143034
Appears in Collections:
Articles; Bioscience Program; Environmental Science and Engineering Program; Center for Desert Agriculture; Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.authorFrusciante, Sarahen
dc.contributor.authorDiretto, Gianfrancoen
dc.contributor.authorBruno, Marken
dc.contributor.authorFerrante, Paolaen
dc.contributor.authorPietrella, Marcoen
dc.contributor.authorPrado-Cabrero, Alfonsoen
dc.contributor.authorRubio-Moraga, Ángela L.en
dc.contributor.authorBeyer, Peter D.en
dc.contributor.authorGómez-Gómez, Lourdesen
dc.contributor.authorAl-Babili, Salimen
dc.contributor.authorGiuliano, Giovannien
dc.date.accessioned2015-08-03T12:06:35Zen
dc.date.available2015-08-03T12:06:35Zen
dc.date.issued2014-08-05en
dc.identifier.issn00278424en
dc.identifier.pmid25097262en
dc.identifier.doi10.1073/pnas.1404629111en
dc.identifier.urihttp://hdl.handle.net/10754/563692en
dc.description.abstractCrocus 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.en
dc.description.sponsorshipWe thank Hansgeorg Ernst for providing the synthetic substrates; Chiara Lico for a gift of N. benthamiana plants and for helpin agroinfiltration experiments; Elena Romano and Emanuela Viaggiu at the Centre of Advanced Microscopy "Patrizia Albertano" for the confocal images; Gaetano Perrotta, Paolo Facella, and Fabrizio Carbone for 454 sequencing; and Alessia Fiore for critical reading of the manuscript. This work was supported by the Italian Ministry of Research (Project "Integrated Knowledge for the Sustainability and Innovation of Italian Agri-Food"), German Research Foundation (Deutsche Forschungsgemeinschaft) Grant AL 892/1-4, the European Union [The development of tools and effective strategies for the optimisation of useful secondary metabolite production in planta, Seventh Framework Programme (FP7) Contract 244348; From discovery to products: A next generation pipeline for the sustainable generation of high-value plant products, FP7 Contract 613153], the King Abdullah University of Science and Technology, the Spanish Ministerio de Ciencia e Innovacion (BIO2009-07803), and the Iberoamerican Network for the Study of Carotenoids as Food Ingredients (112RT0445). S. F. was supported by short-term fellowships of the PlantEngine (FA1006) and Saffronomics (FA1101) European Cooperation in Science and Technology actions.en
dc.publisherProceedings of the National Academy of Sciencesen
dc.relation.urlhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4143034en
dc.subjectSymmetric carotenoid cleavageen
dc.subjectβ-citraurinen
dc.titleNovel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesisen
dc.typeArticleen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
dc.contributor.departmentCenter for Desert Agricultureen
dc.contributor.departmentEnvironmental Science and Engineering Programen
dc.contributor.departmentBioscience Programen
dc.identifier.journalProceedings of the National Academy of Sciencesen
dc.identifier.pmcidPMC4143034en
dc.contributor.institutionItalian National Agency for New Technologies, Energy, and Sustainable Development, Casaccia Research Centre, 00123 Rome, Italyen
dc.contributor.institutionSapienza, University of Rome, 00185 Rome, Italyen
dc.contributor.institutionFaculty of Biology, University of Freiburg, D-79104 Freiburg, Germanyen
dc.contributor.institutionInstituto Botánico, Facultad de Farmacia, Universidad de Castilla-La Mancha, 02071 Albacete, Spainen
kaust.authorPrado-Cabrero, Alfonsoen
kaust.authorAl-Babili, Salimen

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