Identification of bacterial carotenoid cleavage dioxygenase homologues that cleave the interphenyl alpha,beta double bond of stilbene derivatives via a monooxygenase reaction.

Carotenoid cleavage oxygenases (CCOs), which are also referred to as carotenoid cleavage dioxygenases (CCDs) are a new class of nonheme iron-type enzymes that oxidatively cleave double bonds in the conjugated carbon chain of carotenoids. The oxidative cleavage mechanism of these enzymes is not clear, and both monooxygenase and dioxygenase mechanisms have been proposed for different carotenoid cleavage enzymes. CCOs have been described from plants, animals, fungi, and cyanobacteria, but little is known about their distribution and activities in bacteria other than cyanobacteria. We surveyed bacterial genome sequences for CCO homologues and report the characterization of CCO homologues that were identified in Novosphingobium aromaticivorans DSM 12444 (NOV1 and NOV2) and in Bradyrhizobium sp. (BRA-J and BRA-S). In vitro and in vivo assays with carotenoid and stilbene compounds were used to investigate the cleavage activities of the recombinant enzymes. The NOV enzymes cleaved the interphenyl alpha-beta double bond of stilbenes that had an oxygen functional group at the 4' carbon atom (e.g., resveratrol, piceatannol, and rhaponticin) to the corresponding aldehyde products. Carotenoids and apocarotenoids were not substrates for these enzymes. The two homologous enzymes from Bradyrhizobium sp. did not possess carotenoid or stilbene cleavage oxygenase activities, but showed activity with farnesol. To investigate whether the oxidative cleavage of stilbenes proceeds via a monooxygenase or dioxygenase reaction, oxygen-labeling studies were conducted with NOV2. Our labeling studies show that the double-bond cleavage of stilbenes occurs via a monooxygenase reaction mechanism.