title: Oxylipin biosynthesis in spikemoss Selaginella moellendorffii: Molecular cloning and identification of divinyl ether synthases CYP74M1 and CYP74M3.
abstract: Nonclassical P450s of CYP74 family control the secondary conversions of fatty acid hydroperoxides to bioactive oxylipins in plants. At least ten genes attributed to four novel CYP74 subfamilies have been revealed by the recent sequencing of the spikemoss Selaginella moellendorffii Hieron genome. Two of these genes CYP74M1 and CYP74M3 have been cloned in the present study. Both recombinant proteins CYP74M1 and CYP74M3 were active towards the 13(S)-hydroperoxides of α-linolenic and linoleic acids (13-HPOT and 13-HPOD, respectively) and exhibited the activity of divinyl ether synthase (DES). Products were analyzed by gas chromatography-mass spectrometry. Individual oxylipins were purified by HPLC and finally identified by their NMR data, including the (1)H NMR, 2D-COSY, HSQC and HMBC. CYP74M1 (SmDES1) specifically converted 13-HPOT to (11Z)-etherolenic acid and 13-HPOD to (11Z)-etheroleic acid. CYP74M3 (SmDES2) turned 13-HPOT and 13-HPOD mainly to etherolenic and etheroleic acids, respectively. CYP74M1 and CYP74M3 are the first DESs detected in non-flowering plants. The obtained results demonstrate the existence of the sophisticated oxylipin biosynthetic machinery in the oldest taxa of vascular plants.
authors.affiliation: Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan 420111, Russia.
authors.forename: Svetlana S
authors.initials: SS
authors.lastname: Gorina
authors.affiliation: Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan 420111, Russia.
authors.forename: Yana Y
authors.initials: YY
authors.lastname: Toporkova
authors.affiliation: Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan 420111, Russia.
authors.forename: Lucia S
authors.initials: LS
authors.lastname: Mukhtarova
authors.affiliation: Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan 420111, Russia.
authors.forename: Elena O
authors.initials: EO
authors.lastname: Smirnova
authors.affiliation: Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan 420111, Russia.
authors.forename: Ivan R
authors.initials: IR
authors.lastname: Chechetkin
authors.affiliation: Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan 420111, Russia.
authors.forename: Bulat I
authors.initials: BI
authors.lastname: Khairutdinov
authors.affiliation: Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan 420111, Russia.
authors.forename: Yuri V
authors.initials: YV
authors.lastname: Gogolev
authors.affiliation: Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan 420111, Russia. Electronic address: grechkin@kibb.knc.ru.
authors.forename: Alexander N
authors.initials: AN
authors.lastname: Grechkin
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D007700:Kinetics; D008041:Linoleic Acids / Q000378:metabolism; D008042:Linolenic Acids / Q000378:metabolism; D008054:Lipid Peroxides / Q000378:metabolism; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D054883:Oxylipins / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D032503:Selaginellaceae / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D014753:Vinyl Compounds / Q000378:metabolism*
chemical_list: D008041:Linoleic Acids; D008042:Linolenic Acids; D008054:Lipid Peroxides; D054883:Oxylipins; D010940:Plant Proteins; D011994:Recombinant Proteins; D014753:Vinyl Compounds; C038649:13-hydroperoxy-9,11-octadecadienoic acid; C057375:13-hydroperoxy-9,11,15-octadecatrienoic acid; C100195:vinyl ether; D003577:Cytochrome P-450 Enzyme System; C100485:divinyl ether synthase
keywords: CYP74M subfamily; Divinyl ether synthase; Gemmiferous spikemoss (Selaginella moellendorffii Hieron); Molecular cloning; Oxylipins; P450