The carotenase AtCCD1 from Arabidopsis thaliana is a dioxygenase

Apocarotenoids resulting from the oxidative cleavage of carotenoids serve as important signaling and accessory molecules in a variety of biological processes. The enzymes catalyzing these reactions are referred to as carotenases or carotenoid oxygenases. Whether they act according to a monooxygenase mechanism, requiring two oxygens from different sources, or a dioxygenase mechanism is still a topic of controversy. In this study, we utilized the readily available beta-apo-8'-carotenal as a substrate for the heterologously expressed AtCCD1 protein from Arabidopsis thaliana to investigate the oxidative cleavage mechanism of the 9,10 double bond of carotenoids. beta-Ionone and a C-17-dialdehyde were detected as products by gas and liquid chromatography-mass spectrometry as well as NMR analysis. Labeling experiments using H-2 O-18 or O-18(2) showed that the oxygen in the keto-group of beta-ionone is derived solely from molecular dioxygen. When experiments were performed in an O-18(2)-enriched atmosphere, a substantial fraction of the C-17-dialdehyde contained labeled oxygen. The results unambiguously demonstrate a dioxygenase mechanism for the carotenase AtCCD1 from A. thaliana.