Detection of the First Epoxyalcohol Synthase/Allene Oxide Synthase (CYP74 Clan) in the Lancelet (Branchiostoma belcheri, Chordata)

Abstract

The CYP74 clan cytochromes (P450) are key enzymes of oxidative metabolism of polyunsaturated fatty acids in plants, some Proteobacteria, brown and green algae, and Metazoa. The CYP74 enzymes, including the allene oxide synthases (AOSs), hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases (EASs) transform the fatty acid hydroperoxides to bioactive oxylipins. A novel CYP74 clan enzyme CYP440A18 of the Asian (Belcher’s) lancelet (Branchiostoma belcheri, Chordata) was biochemically characterized in the present work. The recombinant CYP440A18 enzyme was active towards all substrates used: linoleate and α-linolenate 9- and 13-hydroperoxides, as well as with eicosatetraenoate and eicosapentaenoate 15-hydroperoxides. The enzyme specifically converted α-linolenate 13-hydroperoxide (13-HPOT) to the oxiranyl carbinol (9Z,11R,12R,13S,15Z)-11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid (EAS product), α-ketol, 12-oxo-13-hydroxy-9,15-octadecadienoic acid (AOS product), and cis-12-oxo-10,15-phytodienoic acid (AOS product) at a ratio of around 35:5:1. Other hydroperoxides were converted by this enzyme to the analogous products. In contrast to other substrates, the 13-HPOT and 15-HPEPE yielded higher proportions of α-ketols, as well as the small amounts of cyclopentenones, cis-12-oxo-10,15-phytodienoic acid and its higher homologue, dihomo-cis-12-oxo-3,6,10,15-phytotetraenoic acid, respectively. Thus, the CYP440A18 enzyme exhibited dual EAS/AOS activity. The obtained results allowed us to ascribe a name “B. belcheri EAS/AOS” (BbEAS/AOS) to this enzyme. BbEAS/AOS is a first CYP74 clan enzyme of Chordata species possessing AOS activity.

Identification and Characterization of Jasmonic Acid Biosynthetic Genes in Salvia miltiorrhiza Bunge

“…Two AOS genes have been reported in barley ( Hordeum vulgare ) [ 46 ] and tomato ( Solanum lycopersicum ) [ 47 , 48 ], with three in potato ( S. tuberosum ) [ 49 ]. AOSs are a class of CYP74 enzymes [ 50 ], which includes divinyl ether synthase (DES) [ 51 ], hydroperoxide lyase (HPL, synonym hemiacetal synthase) [ 52 ], and epoxyalcohol synthase (EAS) [ 53 ]. Interestingly, several CYP74 enzymes possess dual HPL/EAS or triple HPL/EAS/AOS activities [ 54 , 55 ].…”

Detection of Unprecedented CYP74 Enzyme in Mammal: Hydroperoxide Lyase CYP74C44 of the Bat Sturnira hondurensis

“…No CYP74 enzymes have been detected in mammals before. Only non-mammalian metazoans such as lancelets [ 9 , 25 ], sea anemones [ 26 , 27 ], and stony corals [ 9 ] have been shown to possess the CYP74 clan enzymes.…”

Epoxyalcohol Synthase Branch of Lipoxygenase Cascade

“…Thus, EASs are apparently an ancient group of enzymes that appeared before the separation of plants and Metazoa. We identified and cloned the genes of four CYP74-like proteins belonging to the CYP74 clan (not family) from non-plant organisms, namely the brown alga Ectocarpus siliculosus [127], the starlet sea anemone Nematostella vectensis [128,129], and the lancelet Branchiostoma belcheri [130]. CYP5164B1 of E. siliculosus [127] and CYP443D1 of N. vectensis [128] are true EASs, while two other enzymes possess double activities.…”