Alterations of asparagus (Asparagus officinalis L.) divinyl ether synthase (CYP74H2) catalysis by site-directed...

Divinyl ether synthases (DESs) are the enzymes catalyzing the dehydration of fatty acid hydroperoxides to divinyl ether oxylipins. DESs, along with allene oxide synthases (AOSs), hydroperoxide lyases (HPLs), and epoxyalcohol synthases (EASs), are members of the CYP74 clan of unusual cytochromes P450 playing a key role in the lipoxygenase pathway. The present work focuses on the study of the structure-function relationships in Asparagus officinalis DES (AoDES, CYP74H2) via site-directed mutagenesis at some catalytically essential sites. Single mutant forms L106F and L282G retained the DES activity. However, the L106F mutant possessed significant alteration of stereochemical specificity of divinyl ether synthesis compared with WT AoDES. For example, while WT AoDES specifically converted linoleic acid 13(S)-hydroperoxide into (11Z)-etheroleic acid, its yield was significantly reduced by the L106F mutation, whereas etheroleic and (all-E)-etheroleic acids were the major ones. In contrast, the L282G mutation did not significantly affect the (11Z)-etheroleic acid formation. However, the L282G protein produced some additional products like those of HPL and EAS, along with divinyl ethers. The L106F/L282G double mutant protein lost DES activity. It converted α-linolenic 9- and 13-hydroperoxides into HPL chain cleavage products. At the same time, this mutant efficiently converted the linoleic acid 9-hydroperoxide into diol, 9,14-dihydroxy-10,12-octadecadienoic acid, presumably via the hydrolysis of the short-lived epoxydiene, 9,10-epoxy-11,13-octadecadienoic acid. Furthermore, the L106F/L282G/Q343P triple mutant showed AOS activity alongside DES. The appearance of EAS and HPL catalysis, as well as the biosynthesis of 9,14-epoxydiene via short-lived epoxydiene via site-directed mutagenesis in the catalytically relevant domains of DES, was demonstrated for the first time.