- Detection and molecular cloning of CYP74Q1 gene: Identification of Ranunculus acris leaf divinyl ether synthase
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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.
- Gorina, Svetlana S.,Toporkova, Yana Y.,Mukhtarova, Lucia S.,Chechetkin, Ivan R.,Khairutdinov, Bulat I.,Gogolev, Yuri V.,Grechkin, Alexander N.
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- The CYP74B and CYP74D divinyl ether synthases possess a side hydroperoxide lyase and epoxyalcohol synthase activities that are enhanced by the site-directed mutagenesis
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The CYP74 family of cytochromes P450 includes four enzymes of fatty acid hydroperoxide metabolism: allene oxide synthase (AOS), hydroperoxide lyase (HPL), divinyl ether synthase (DES), and epoxyalcohol synthase (EAS). The present work is concerned with catalytic specificities of three recombinant DESs, namely, the 9-DES (LeDES, CYP74D1) of tomato (Solanum lycopersicum), 9-DES (NtDES, CYP74D3) of tobacco (Nicotiana tabacum), and 13-DES (LuDES, CYP74B16) of flax (Linum usitatissimum), as well as their alterations upon the site-directed mutagenesis. Both LeDES and NtDES converted 9-hydroperoxides of linoleic and α?linolenic acids to divinyl ethers colneleic and colnelenic acids (respectively) with only minorities of HPL and EAS products. In contrast, LeDES and NtDES showed low efficiency towards the linoleate 13-hydroperoxide, affording only the low yield of epoxyalcohols. LuDES exhibited mainly the DES activity towards α?linolenate 13-hydroperoxide (preferred substrate), and HPL activity towards linoleate 13-hydroperoxide, respectively. In contrast, LuDES converted 9-hydroperoxides primarily to the epoxyalcohols. The F291V and A287G mutations within the I-helix groove region (SRS-4) of LuDES resulted in the loss of DES activity and the acquirement of the epoxyalcohol synthase activity. Thus, the studied enzymes exhibited the versatility of catalysis and its qualitative alterations upon the site-directed mutagenesis.
- Gorina, Svetlana S.,Grechkin, Alexander N.,Iljina, Tatiana M.,Mukhtarova, Lucia S.,Smirnova, Elena O.,Toporkova, Yana Y.
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- Detection and identification of complex oxylipins in meadow buttercup (Ranunculus acris) leaves
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Screening of linolipins, i.e. galactolipids containing esterified residues of divinyl ether oxylipins, in the leaves of several higher plants revealed the presence of these complex oxylipins in the meadow buttercup leaves. The rapid accumulation of linoli
- Chechetkin, Ivan R.,Blufard, Alexander S.,Yarin, Andrey Y.,Fedina, Evgenia O.,Khairutdinov, Bulat I.,Grechkin, Alexander N.
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- A lipoxygenase-divinyl ether synthase pathway in flax (Linum usitatissimum L.) leaves
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Incubation of linoleic acid with an enzyme preparation from leaves of flax (Linum usitatissimum L.) led to the formation of a divinyl ether fatty acid, i.e. (9Z,11E,1′Z)-12-(1′-hexenyloxy)-9,11-dodecadienoic [(ω5Z)-etheroleic] acid, as well as smaller amounts of 13-hydroxy-9(Z),11(E)-octadecadienoic acid. The 13-hydroperoxide of linoleic acid afforded the same set of products, whereas incubations of α-linolenic acid and its 13-hydroperoxide afforded the divinyl ether (9Z,11E,1′Z,3′Z)-12-(1′,3′-hexadienyloxy)-9,11-dodecadienoic [(ω5Z)-etherolenic] as the main product. Identification of both divinyl ethers was substantiated by their UV, mass-, 1H NMR and COSY spectral data. In addition to the 13-lipoxygenase and divinyl ether synthase activities demonstrated by these results, flax leaves also contained allene oxide synthase activity as judged by the presence of endogenously formed (15Z)-cis-12-oxo-10,15-phytodienoic acid in all incubations.
- Chechetkin, Ivan R.,Blufard, Alexander,Hamberg, Mats,Grechkin, Alexander N.
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p. 2008 - 2015
(2008/12/21)
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