title: Detection and molecular cloning of CYP74Q1 gene: identification of Ranunculus acris leaf divinyl ether synthase.
abstract: Enzymes of the CYP74 family, including the divinyl ether synthase (DES), play important roles in plant cell signalling and defence. The potent DES activities have been detected before in the leaves of the meadow buttercup (Ranunculus acris L.) and few other Ranunculaceae species. The nature of these DESs and their genes remained unrevealed. The PCR with degenerate primers enabled to detect the transcript of unknown P450 gene assigned as CYP74Q1. Besides, two more CYP74Q1 isoforms with minimal sequence variations have been found. The full length recombinant CYP74Q1 protein was expressed in Escherichia coli. The preferred substrates of this enzyme are the 13-hydroperoxides of α-linolenic and linoleic acids, which are converted to the divinyl ether oxylipins (ω5Z)-etherolenic acid, (9Z,11E)-12-[(1'Z,3'Z)-hexadienyloxy]-9,11-dodecadienoic acid, and (ω5Z)-etheroleic acid, (9Z,11E)-12-[(1'Z)-hexenyloxy]-9,11-dodecadienoic acid, respectively, as revealed by the data of mass spectrometry, NMR and UV spectroscopy. Thus, CYP74Q1 protein was identified as the R. acris DES (RaDES), a novel DES type and the opening member of new CYP74Q subfamily.
authors.affiliation: Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, 420111 Kazan, 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, 420111 Kazan, 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, 420111 Kazan, 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, 420111 Kazan, 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, 420111 Kazan, 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, 420111 Kazan, 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, 420111 Kazan, Russia. Electronic address: grechkin@mail.knc.ru.
authors.forename: Alexander N
authors.initials: AN
authors.lastname: Grechkin
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007527:Isoenzymes / Q000145:classification / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008041:Linoleic Acids / Q000378:metabolism; D008969:Molecular Sequence Data; D054883:Oxylipins / Q000378:metabolism; D010802:Phylogeny; D018515:Plant Leaves / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D031947:Ranunculus / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D017962:alpha-Linolenic Acid / Q000378:metabolism
chemical_list: D017931:DNA Primers; D007527:Isoenzymes; D008041:Linoleic Acids; D054883:Oxylipins; D010940:Plant Proteins; D011994:Recombinant Proteins; D017962:alpha-Linolenic Acid; D003577:Cytochrome P-450 Enzyme System; C100485:divinyl ether synthase
keywords: CYP74Q1; Divinyl ether synthase; Meadow buttercup (Ranunculus acris L.); Molecular cloning; Oxylipins; P450