Svetlana S. Gorina

Structure–function relationship in the CYP74 family: Conversion of divinyl ether synthases into allene oxide synthases by site-directed mutagenesis

Non‐classical P450s of CYP74 family control several enzymatic conversions of fatty acid hydroperoxides to bioactive oxylipins in plants, some invertebrates and bacteria. The family includes two dehydrases, namely allene oxide synthase (AOS) and divinyl ether synthase (DES), and two isomerases, hydroperoxide lyase (HPL) and epoxyalcohol synthase. To study the interconversion of different CYP74 enzymes, we prepared the mutant forms V379F and E292G of tobacco (CYP74D3) and flax (CYP74B16) divinyl ether synthases (DESs), respectively. In contrast to the wild type (WT) enzymes, both mutant forms lacked DES activity. Instead, they produced the typical AOS products, α‐ketols and (in the case of the flax DES mutant) 12‐oxo‐10,15‐phytodienoic acid. This is the first demonstration of DES into AOS conversions caused by single point mutations.

Isolation and structure elucidation of linolipins C and D, complex oxylipins from flax leaves

Two complex oxylipins (linolipins C and D) were isolated from the leaves of flax plants inoculated with phytopathogenic bacteria Pectobacterium atrosepticum. Their structures were elucidated based on UV, MS and NMR spectroscopic data. Both oxylipins were identified as digalactosyldiacylglycerol (DGDG) molecular species. Linolipin C contains one residue of divinyl ether (ω5Z)-etherolenic acid and one α-linolenate residue at sn-1 and sn-2 positions, respectively. Linolipin D possesses two (ω5Z)-etherolenic acid residues at both sn-1 and sn-2 positions. The rapid formation (2-30min) of linolipins C and D alongside with linolipins A and B occurred in the flax leaves upon their damage by freezing-thawing.

Novel Allene Oxide Synthase Products Formed via Favorskii-Type Rearrangement: Mechanistic Implications for 12-Oxo-10,15-phytodienoic Acid Biosynthesis

The allene oxide synthase (AOS) pathway is widespread in plants. Its products, such as cyclopentenone 12‐oxo‐10,15‐phytodienoic acid (12‐oxo‐PDA) and related jasmonates, play important biological roles in plants. We found that 12‐oxo‐PDA in some plant tissues co‐occur with an unknown minor oxylipin 1. In vitro incubations of AOSs with α‐linolenic acid 13(S)‐hydroperoxide reliably afforded 1 along with 12‐oxo‐PDA and α‐ketol. A similar oxylipin 3 was formed during the AOS conversions of γ‐linolenic acid 9(S)‐hydroperoxide. Linoleic acid hydroperoxides formed neither products similar to 1 and 3 nor cyclopentenones. Oxylipins 1 and 3 were purified and identified as the products of Favorskii‐type rearrangement, (2′Z,4Z)‐2‐(2′‐pentenyl)‐4‐tridecene‐1,13‐dioic acid and (2′Z,4Z)‐2‐(2′‐octenyl)‐4‐decene‐1,10‐dioic acid, respectively. Detection of Favorskii products 1 and 3 demonstrates that cyclopropanones are short‐lived AOS products along with allene oxides. The observed parallels between the Favorskii product 1 and 12‐oxo‐PDA formation suggests that cyclopropanone is either a byproduct or a precursor of 12‐oxo‐PDA.

Oxylipin biosynthesis in spikemoss Selaginella moellendorffii: Molecular cloning and identification of divinyl ether synthases CYP74M1 and CYP74M3.

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.

Stereospecific biosynthesis of (9S,13S)-10-oxo-phytoenoic acid in young maize roots.

Profiling of oxylipins from young maize roots revealed complex patterns of products mainly originating from the combined actions of 9- and 13-lipoxygenases and allene oxide synthase (AOS). A distinctive feature was the high content of the cyclopentenone 10-oxo-11-phytoenoic acid (10-oxo-PEA). Incubations with [1-14C]linoleic acid led to the formation of the α-ketols 13-hydroxy-12-oxo-9-octadecenoic acid and 9-hydroxy-10-oxo-12-octadecenoic acid as well as the cyclopentenones 12-oxo-10-phytoenoic acid (12-oxo-PEA) and 10-oxo-PEA in a ratio of 10:2:1:3. Chiral phase radio-HPLC showed that the labeled 10-oxo-PEA was mainly (93%) due to the 9S,13S-enantiomer, whereas 12-oxo-PEA was racemic. Recombinant maize AOS CYP74A19 (ZmAOS2) converted linoleic acid 9(S)-hydroperoxide (9-HPOD) into an allene oxide, 9,10-epoxy-10,12-octadecadienoic acid (9,10-EOD), which did not undergo cyclization but was solely hydrolyzed into the α-ketol. A cyclase activity promoting the conversion of 9,10-EOD into (9S,13S)-10-oxo-PEA was detected in the 10(5)×g supernatant prepared by differential centrifugation of the maize root homogenate. The data obtained suggested the existence of a new type of allene oxide cyclase, which is active towards an allene oxide formed from a 9-lipoxygenase-derived hydroperoxide.

Detection of the first higher plant epoxyalcohol synthase: Molecular cloning and characterisation of the CYP74M2 enzyme of spikemoss Selaginella moellendorffii

The CYP74M2 gene of a model plant, the spikemoss Selaginella moellendorffii Hieron, was cloned and the catalytic properties of corresponding recombinant protein were studied. The recombinant CYP74M2 protein was active towards 13-hydroperoxides of linoleic and a-linolenic acids (13-HPOD and 13-HPOT, respectively). In contrast to previously studied CYP74M1 and CYP74M3, which possessed the divinyl ether synthase activity, CYP74M2 behaved as a dedicated epoxyalcohol synthase (EAS). For instance, the 13-HPOD was converted to three epimeric oxiranyl carbinols 1-3 (formed at a ratio ca. 4:2:1), namely the (11R,12S,13S), (11R,12R, 13S), and (11S,12S,13S) epimers of (9Z)-11-hydroxy-12,13-epoxy-9-octadecenoic acid. Besides these products, a minority of oxiranyl vinyl carbinols like (10E)-11-hydroxy-12,13-epoxy-9-octadecenoic acid was formed. The 13-HPOT conversion by CYP74M2 afforded two stereoisomers of 11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid. Individual oxylipins were purified by HPLC and finally identified by their NMR data, including the 1H-NMR, 2D-COSY, HSQC, and HMBC. Thus, the CYP74M2 is the dedicated epoxyalcohol synthase. To our knowledge, no enzymes of this type have been detected in higher plants yet.

Antimicrobial Activity of Geometric Isomers of Etherolenic Acid—the Products of Plant Lipoxygenase Cascade

Data on the influence of the double bond geometry on the antimicrobial properties of different isomers of etherolenic acid against phytopathogenic bacteria are presented. (ω5Z)-Etherolenic acid possesses bactericidal properties against Xanthomonas campestris ssp. vesicatoria, Pseudomonas syringae ssp. tomato, Pectobacterium atrosepticum SCRI1043; the etherolenic and (11Z)-etherolenic acids possess only bacteriostatic properties.