TY - JOUR
T1 - Identification of the gene responsible for torulene cleavage in the Neurospora carotenoid pathway
AU - Saelices, Lorena
AU - Youssar, Loubna
AU - Holdermann, Iris
AU - Al-Babili, Salim
AU - Avalos, Javier
N1 - Funding Information:
Acknowledgments We thank Dr. T. J. Schmidhauser for laboratory facilities in the search of the color mutants, Dr. Hansgeorg Ernst (BASF) for providing the apocarotenoids, Dr. Jorge Mayer for proofreading the manuscript, and Dr. Peter Beyer for valuable discussions. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) Grant AL892-3, HarvestPlus (www.harvestplus.org) and the Spanish Government (Ministerio de Ciencia y Tecnología, projects BIO2003-01548 and BIO2006-01323).
PY - 2007/11
Y1 - 2007/11
N2 - Torulene, a C40 carotene, is the precursor of the end product of the Neurospora carotenoid pathway, the C35 xanthophyll neurosporaxanthin. Torulene is synthesized by the enzymes AL-2 and AL-1 from the precursor geranylgeranyl diphosphate and then cleaved by an unknown enzyme into the C35 apocarotenoid. In general, carotenoid cleavage reactions are catalyzed by carotenoid oxygenases. Using protein data bases, we identified two putative carotenoid oxygenases in Neurospora, named here CAO-1 and CAO-2. A search for novel mutants of the carotenoid pathway in this fungus allowed the identification of two torulene-accumulating strains, lacking neurosporaxanthin. Sequencing of the cao-2 gene in these strains revealed severe mutations, pointing to a role of CAO-2 in torulene cleavage. This was further supported by the identical phenotype found upon targeted disruption of cao-2. The biological function was confirmed by in vitro assays using the purified enzyme, which cleaved torulene to produce β-apo-4′-carotenal, the corresponding aldehyde of neurosporaxanthin. The specificity of CAO-2 was shown by the lack of γ-carotene-cleaving activity in vitro. As predicted for a structural gene of the carotenoid pathway, cao-2 mRNA was induced by light in a WC-1 and WC-2 dependent manner. Our data demonstrate that CAO-2 is the enzyme responsible for the oxidative cleavage of torulene in the neurosporaxanthin biosynthetic pathway.
AB - Torulene, a C40 carotene, is the precursor of the end product of the Neurospora carotenoid pathway, the C35 xanthophyll neurosporaxanthin. Torulene is synthesized by the enzymes AL-2 and AL-1 from the precursor geranylgeranyl diphosphate and then cleaved by an unknown enzyme into the C35 apocarotenoid. In general, carotenoid cleavage reactions are catalyzed by carotenoid oxygenases. Using protein data bases, we identified two putative carotenoid oxygenases in Neurospora, named here CAO-1 and CAO-2. A search for novel mutants of the carotenoid pathway in this fungus allowed the identification of two torulene-accumulating strains, lacking neurosporaxanthin. Sequencing of the cao-2 gene in these strains revealed severe mutations, pointing to a role of CAO-2 in torulene cleavage. This was further supported by the identical phenotype found upon targeted disruption of cao-2. The biological function was confirmed by in vitro assays using the purified enzyme, which cleaved torulene to produce β-apo-4′-carotenal, the corresponding aldehyde of neurosporaxanthin. The specificity of CAO-2 was shown by the lack of γ-carotene-cleaving activity in vitro. As predicted for a structural gene of the carotenoid pathway, cao-2 mRNA was induced by light in a WC-1 and WC-2 dependent manner. Our data demonstrate that CAO-2 is the enzyme responsible for the oxidative cleavage of torulene in the neurosporaxanthin biosynthetic pathway.
KW - CAO-2
KW - Carotenoid oxygenase
KW - Light regulation
KW - Neurosporaxanthin
KW - β-apo-4′-carotenal
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U2 - 10.1007/s00438-007-0269-2
DO - 10.1007/s00438-007-0269-2
M3 - Article
C2 - 17610084
AN - SCOPUS:35248846478
SN - 1617-4615
VL - 278
SP - 527
EP - 537
JO - Molecular Genetics and Genomics
JF - Molecular Genetics and Genomics
IS - 5
ER -