Functional characterization of FaCCD1: a carotenoid cleavage dioxygenase from strawberry involved in lutein degradation during fruit ripening.
A gene encoding a carotenoid cleavage dioxygenase class 1 enzyme (FaCCD1) was identified among a strawberry fruit expressed sequence tag collection. The full-length cDNA was isolated, and the expression profiles along fruit receptacle development and ripening, determined by quantitative real time polymerase chain reaction, showed that FaCCD1 is a ripening-related gene that reaches its maximal level of expression in the red fully ripe stage. FaCCD1 was expressed in Escherichia coli, and the products formed by the recombinant protein through oxidative cleavage of carotenoids were identified by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry analyses. The FaCCD1 protein cleaves zeaxanthin, lutein, and beta-apo-8'-carotenal in vitro. Although beta-carotene is not a good substrate for FaCCD1 in vitro, the expression of FaCCD1 in an engineered carotenoid-producing E. coli strain caused the degradation of beta-carotene in vivo. Additionally, the carotenoid profile in strawberry was analyzed by high-performance liquid chromatography-photodiode detection, and a correlation between the increase of the expression level of FaCCD1 during ripening and the decrease of the lutein content suggests that lutein could constitute the main natural substrate of FaCCD1 activity in vivo.
García-Limones
Carmen
C
Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, 14071 Córdoba, Spain. bb1mublj@uco.es
Schnäbele
Kerstin
K
Blanco-Portales
Rosario
R
Luz Bellido
M
M
Caballero
Jose Luis
JL
Schwab
Wilfried
W
Muñoz-Blanco
Juan
J
D003001:Cloning, Molecular; D018744:DNA, Plant / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D049308:Dioxygenases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D031985:Fragaria / Q000201:enzymology* / Q000235:genetics; D005638:Fruit / Q000201:enzymology* / Q000254:growth & development*; D015870:Gene Expression; D014975:Lutein / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017385:Sequence Homology; D013379:Substrate Specificity
D018744:DNA, Plant; D011994:Recombinant Proteins; D049308:Dioxygenases; C431939:carotenoid cleavage dioxygenase 1; D014975:Lutein