earticle

영어

Carotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preference of the CCD NSC3 of Nostoc sp. PCC 7120 in vivo and in vitro using natural and non-natural structures of carotenoids. NSC3 cleaved β-apo-8′-carotenal at 3 specific central double bonds in the chromophore, C13-C14, C15-C15’, and C13’-C14’, revealing a unique cleavage pattern. The modular expression of carotenoid pathway enzymes in Escherichia coli provides NSC3 with natural and non-natural carotenoid structures in vivo and in vitro. NSC3 used natural C30 carotenoids, 4,4’-diaponeurosporene, 4,4’-diaponeurosporen-4’-al, and 4,4’-diaponeurosporen-4’-oic acid as substrates to generate structurally novel cleavage products, apo-14’-diaponeurosporenal, apo-13’- diaponeurosporenal, and apo-10’-diaponeurosporenal. Furthermore, NSC3 catalyzed cleavage activity on non-natural C30 carotenoids, 4,4’- diapotorulene and 4,4’-diapotorulen-4’-al to produce apo-14’- diapotorulenal and apo-10’-diapotorulenal, respectively. Use of carotenoids of natural or non-natural structures which are produced by synthetic modules could provide valuable information for understanding other CCDs’ cleavage reaction or substrate preference in vivo and in vitro.