Epoxyalcohol Synthase Branch of Lipoxygenase Cascade.

Oxylipins are one of the most important classes of bioregulators, biosynthesized through the oxidative metabolism of unsaturated fatty acids in various aerobic organisms. Oxylipins are bioregulators that maintain homeostasis at the cellular and organismal levels. The most important oxylipins are mammalian eicosanoids and plant octadecanoids. In plants, the main source of oxylipins is the lipoxygenase cascade, the key enzymes of which are nonclassical cytochromes P450 of the CYP74 family, namely allene oxide synthases (AOSs), hydroperoxide lyases (HPLs), and divinyl ether synthases (DESs). The most well-studied plant oxylipins are jasmonates (AOS products) and traumatin and green leaf volatiles (HPL products), whereas other oxylipins remain outside of the focus of researchers' attention. Among them, there is a large group of epoxy hydroxy fatty acids (epoxyalcohols), whose biosynthesis has remained unclear for a long time. In 2008, the first epoxyalcohol synthase of lancelet Branchiostoma floridae, BfEAS (CYP440A1), was discovered. The present review collects data on EASs discovered after BfEAS and enzymes exhibiting EAS activity along with other catalytic activities. This review also presents the results of a study on the evolutionary processes possibly occurring within the P450 superfamily as a whole.

CYP74B34 Enzyme from Carrot (Daucus carota) with a Double Hydroperoxide Lyase/Epoxyalcohol Synthase Activity: Identification and Biochemical Properties.

The lipoxygenase cascade in plants is a source of oxylipins (oxidized fatty acid derivatives), which play an important role in regulatory processes and formation of plant response to stress factors. Some of the most common enzymes of the lipoxygenase cascade are 13-specific hydroperoxide lyases (HPLs, also called hemiacetal synthases) of the CYP74B subfamily. In this work, we identified and cloned the CYP74B34 gene from carrot (Daucus carota L.) and described the biochemical properties of the corresponding recombinant enzyme. The CYP74B34 enzyme was active towards 9- and 13-hydroperoxides of linoleic (9-HPOD and 13-HPOD, respectively) and α-linolenic (9-HPOT and 13-HPOT, respectively) acids. CYP74B34 specifically converted 9-HPOT and 13-HPOT into aldo acids (HPL products). The transformation of 13-HPOD led to the formation of aldo acids and epoxyalcohols [products of epoxyalcohol synthase (EAS) activity] as major and minor products, respectively. At the same time, conversion of 9-HPOD resulted in the formation of epoxyalcohols as the main products and aldo acids as the minor ones. Therefore, CYP74B34 is the first enzyme with a double HPL/EAS activity described in carrot. The presence of these catalytic activities was confirmed by analysis of the oxylipin profiles for the roots from young seedlings and mature plants. In addition, we substituted amino acid residues in one of the catalytically essential sites of the CYP74B34 and CYP74B33 proteins and investigated the properties of the obtained mutant enzymes.

Recombinant Soybean Lipoxygenase 2 (GmLOX2) Acts Primarily as a ω6(S)-Lipoxygenase.

The lipoxygenase (LOX) cascade is a source of bioactive oxylipins that play a regulatory role in plants, animals, and fungi. Soybean (Glycine max (L.) Merr.) LOXs are the classical models for LOX research. Progress in genomics has uncovered a large diversity of GmLOX isoenzymes. Most of them await biochemical investigations. The catalytic properties of recombinant soybean LOX2 (GmLOX2) are described in the present work. The GmLOX2 gene has been cloned before, but only for nucleotide sequencing, while the recombinant protein was not prepared and studied. In the present work, the recombinant GmLOX2 behavior towards linoleic, α-linolenic, eicosatetraenoic (20:4), eicosapentaenoic (20:5), and hexadecatrienoic (16:3) acids was examined. Linoleic acid was a preferred substrate. Oxidation of linoleic acid afforded 94% optically pure (13S)-hydroperoxide and 6% racemic 9-hydroperoxide. GmLOX2 was less active on other substrates but possessed an even higher degree of regio- and stereospecificity. For example, it converted α-linolenic acid into (13S)-hydroperoxide at about 98% yield. GmLOX2 showed similar specificity towards other substrates, producing (15S)-hydroperoxides (with 20:4 and 20:5) or (11S)-hydroperoxide (with 16:3). Thus, the obtained data demonstrate that soybean GmLOX2 is a specific (13S)-LOX. Overall, the catalytic properties of GmLOX2 are quite similar to those of GmLOX1, but pH is optimum.

Discovery of α-Linolenic Acid 16(S)-Lipoxygenase: Cucumber (Cucumis sativus L.) Vegetative Lipoxygenase 3.

The GC-MS profiling of the endogenous oxylipins (Me/TMS) from cucumber (Cucumis sativus L.) leaves, flowers, and fruit peels revealed a remarkable abundance of 16-hydroxy-9,12,14-octadecatrienoic acid (16-HOT). Incubations of homogenates from these organs with α-linolenic acid yielded 16(S)-hydroperoxide (16-HPOT) as a predominant product. Targeted proteomic analyses of these tissues revealed the presence of several highly homologous isoforms of the putative "9S-lipoxygenase type 6". One of these isoenzymes (CsLOX3, an 877 amino acid polypeptide) was prepared by heterologous expression in E. coli and exhibited 16(S)- and 13(S)-lipoxygenase activity toward α-linolenic and linoleic acids, respectively. Furthermore, α-linolenate was a preferred substrate. The molecular structures of 16(S)-HOT and 16(S)-HPOT (Me or Me/TMS) were unequivocally confirmed by the mass spectral data, 1H-NMR, 2D 1H-1H-COSY, TOCSY, HMBC, and HSQC spectra, as well as enantiomeric HPLC analyses. Thus, the vegetative CsLOX3, biosynthesizing 16(S)-HPOT, is the first 16(S)-LOX and ω3-LOX ever discovered. Eicosapentaenoic and hexadecatrienoic acids were also specifically transformed to the corresponding ω3(S)-hydroperoxides by CsLOX3.

Distinct Mechanistic Behaviour of Tomato CYP74C3 and Maize CYP74A19 Allene Oxide Synthases: Insights from Trapping Experiments and Allene Oxide Isolation.

The product specificity and mechanistic peculiarities of two allene oxide synthases, tomato LeAOS3 (CYP74C3) and maize ZmAOS (CYP74A19), were studied. Enzymes were vortexed with linoleic acid 9-hydroperoxide in a hexane-water biphasic system (20-60 s, 0 °C). Synthesized allene oxide (9,10-epoxy-10,12-octadecadienoic acid; 9,10-EOD) was trapped with ethanol. Incubations with ZmAOS produced predominantly 9,10-EOD, which was converted into an ethanolysis product, (12Z)-9-ethoxy-10-oxo-12-octadecenoic acid. LeAOS3 produced the same trapping product and 9(R)-α-ketol at nearly equimolar yields. Thus, both α-ketol and 9,10-EOD appeared to be kinetically controlled LeAOS3 products. NMR data for 9,10-EOD (Me) preparations revealed that ZmAOS specifically synthesized 10(E)-9,10-EOD, whereas LeAOS3 produced a roughly 4:1 mixture of 10(E) and 10(Z) isomers. The cyclopentenone cis-10-oxo-11-phytoenoic acid (10-oxo-PEA) and the Favorskii-type product yields were appreciable with LeAOS3, but dramatically lower with ZmAOS. The 9,10-EOD (free acid) kept in hexane transformed into macrolactones but did not cyclize. LeAOS3 catalysis is supposed to produce a higher proportion of oxyallyl diradical (a valence tautomer of allene oxide), which is a direct precursor of both cyclopentenone and cyclopropanone. This may explain the substantial yields of cis-10-oxo-PEA and the Favorskii-type product (via cyclopropanone) with LeAOS3. Furthermore, 10(Z)-9,10-EOD may be produced via the reverse formation of allene oxide from oxyallyl diradical.

Differential modulation of the lipoxygenase cascade during typical and latent Pectobacterium atrosepticum infections.

BACKGROUND AND AIMS: Plant diseases caused by Pectobacterium atrosepticum are often accompanied by extensive rot symptoms. In addition, these bacteria are able to interact with host plants without causing disease for long periods, even throughout several host plant generations. There is, to date, no information on the comparative physiology/biochemistry of symptomatic and asymptomatic plant-P. atrosepticum interactions. Typical (symptomatic) P. atrosepticum infections are associated with the induction of plant responses mediated by jasmonates, which are one of the products of the lipoxygenase cascade that gives origin to many other oxylipins with physiological activities. In this study, we compared the functioning of the lipoxygenase cascade following typical and latent (asymptomatic) infections to gain better insight into the physiological basis of the asymptomatic and antagonistic coexistence of plants and pectobacteria. METHODS: Tobacco plants were mock-inoculated (control) or infected with the wild type P. atrosepticum (typical infection) or its coronafacic acid-deficient mutant (latent infection). The expression levels of the target lipoxygenase cascade-related genes were assessed by Illumina RNA sequencing. Oxylipin profiles were analysed by GC-MS. With the aim of revising the incorrect annotation of one of the target genes, its open reading frame was cloned to obtain the recombinant protein, which was further purified and characterized using biochemical approaches. KEY RESULTS: The obtained data demonstrate that when compared to the typical infection, latent asymptomatic P. atrosepticum infection is associated with (and possibly maintained due to) decreased levels of 9-lipoxygenase branch products and jasmonic acid and increased level of cis-12-oxo-10,15-phytodienoic acid. The formation of 9-oxononanoic acid and epoxyalcohols in tobacco plants was based on the identification of the first tobacco hydroperoxide lyase (HPL) with additional epoxyalcohol synthase (EAS) activity. CONCLUSIONS: Our results contribute to the hypothesis of the oxylipin signature, indicating that different types of plant interactions with a particular pathogen are characterized by the different oxylipin profiles of the host plant. In addition, the tobacco LOC107825278 gene was demonstrated to encode an NtHPL (CYP74C43) enzyme yielding volatile aldehydes and aldoacids (HPL products) as well as oxiranyl carbinols (EAS products).

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

The genome of the neotropical fruit bat Sturnira hondurensis was recently sequenced, revealing an unexpected gene encoding a plant-like protein, CYP74C44, which shares ca. 90% sequence identity with the putative CYP74C of Populus trichocarpa. The preparation and properties of the recombinant CYP74C44 are described in the present work. The CYP74C44 enzyme was found to be active against the 13- and 9-hydroperoxides of linoleic and α-linolenic acids (13-HPOD, 13-HPOT, 9-HPOD, and 9-HPOT, respectively), as well as the 15-hydroperoxide of eicosapentaenoic acid (15-HPEPE). All substrates studied were specifically transformed into chain cleavage products that are typical for hydroperoxide lyases (HPLs). The HPL chain cleavage reaction was validated by the identification of NaBH4-reduced products (Me/TMS) of 15-HPEPE and 13- and 9-hydroperoxides as (all-Z)-14-hydroxy-5,8,11-tetradecatrienoic, (9Z)-12-hydroxy-9-dodecenoic, and 9-hydroxynonanoic acids (Me/TMS), respectively. Thus, CYP74C44 possessed the HPL activity that is typical for the CYP74C subfamily proteins.

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

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.

Double function hydroperoxide lyases/epoxyalcohol synthases (CYP74C) of higher plants: identification and conversion into allene oxide synthases by site-directed mutagenesis.

The CYP74C subfamily of fatty acid hydroperoxide transforming enzymes includes hydroperoxide lyases (HPLs) and allene oxide synthases (AOSs). This work reports a new facet of the putative CYP74C HPLs. Initially, we found that the recombinant CYP74C13_MT (Medicago truncatula) behaved predominantly as the epoxyalcohol synthase (EAS) towards the 9(S)-hydroperoxide of linoleic acid. At the same time, the CYP74C13_MT mostly possessed the HPL activity towards the 13(S)-hydroperoxides of linoleic and α-linolenic acids. To verify whether this dualistic behaviour of CYP74C13_MT is occasional or typical, we also examined five similar putative HPLs (CYP74C). These were CYP74C4_ST (Solanum tuberosum), CYP74C2 (Cucumis melo), CYP74C1_CS and CYP74C31 (both of Cucumis sativus), and CYP74C13_GM (Glycine max). All tested enzymes behaved predominantly as EAS toward 9-hydroperoxide of linoleic acid. Oxiranyl carbinols such as (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acids were the major EAS products. Besides, the CYP74C31 possessed an additional minor 9-AOS activity. The mutant forms of CYP74C13_MT, CYP74C1_CS, and CYP74C31 with substitutions at the catalytically essential domains, namely the "hydroperoxide-binding domain" (I-helix), or the SRS-1 domain near the N-terminus, showed strong AOS activity. These HPLs to AOSs conversions were observed for the first time. Until now a large part of CYP74C enzymes has been considered as 9/13-HPLs. Notwithstanding, these results show that all studied putative CYP74C HPLs are in fact the versatile HPL/EASs that can be effortlessly mutated into specific AOSs.

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.

Epoxyalcohol synthase of Ectocarpus siliculosus. First CYP74-related enzyme of oxylipin biosynthesis in brown algae.

Enzymes of CYP74 family play the central role in the biosynthesis of physiologically important oxylipins in land plants. Although a broad diversity of oxylipins is known in the algae, no CYP74s or related enzymes have been detected in brown algae yet. Cloning of the first CYP74-related gene CYP5164B1 of brown alga Ectocarpus siliculosus is reported in present work. The recombinant protein was incubated with several fatty acid hydroperoxides. Linoleic acid 9-hydroperoxide (9-HPOD) was the preferred substrate, while linoleate 13-hydroperoxide (13-HPOD) was less efficient. α-Linolenic acid 9- and 13-hydroperoxides, as well as eicosapentaenoic acid 15-hydroperoxide were inefficient substrates. Both 9-HPOD and 13-HPOD were converted into epoxyalcohols. For instance, 9-HPOD was turned primarily into (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acid. Both epoxide and hydroxyl oxygen atoms of the epoxyalcohol were incorporated mostly from [18O2]9-HPOD. Thus, the enzyme exhibits the activity of epoxyalcohol synthase (EsEAS). The results show that the EsEAS isomerizes the hydroperoxides into epoxyalcohols via epoxyallylic radical, a common intermediate of different CYP74s and related enzymes. EsEAS can be considered as an archaic prototype of CYP74 family enzymes.

Identification of CYP443D1 (CYP74 clan) of Nematostella vectensis as a first cnidarian epoxyalcohol synthase and insights into its catalytic mechanism.

The CYP74 clan enzymes are responsible for the biosynthesis of numerous bioactive oxylipins in higher plants, some Proteobacteria, brown and green algae, and Metazoa. A novel putative CYP74 clan gene CYP443D1 of the starlet sea anemone (Nematostella vectensis, Cnidaria) has been cloned, and the properties of the corresponding recombinant protein have been studied in the present work. The recombinant CYP443D1 was incubated with the 9- and 13-hydroperoxides of linoleic and α-linolenic acids (9-HPOD, 13-HPOD, 9-HPOT, and 13-HPOT, respectively), as well as with the 9-hydroperoxide of γ-linolenic acid (γ-9-HPOT) and 15-hydroperoxide of eicosapentaenoic acid (15-HPEPE). The enzyme was active towards all C18-hydroperoxides with some preference to 9-HPOD. In contrast, 15-HPEPE was a poor substrate. The CYP443D1 specifically converted 9-HPOD into the oxiranyl carbinol 1, (9S,10R,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acid. Both 18O atoms from [18O2-hydroperoxy]9-HPOD were virtually quantitatively incorporated into product 1. Thus, the CYP443D1 exhibited epoxyalcohol synthase (EAS) activity. The 18O labelling data demonstrated that the reaction mechanism included three sequential steps: (1) hydroperoxyl homolysis, (2) oxy radical rearrangement into epoxyallylic radical, (3) hydroxyl rebound, resulting in oxiranyl carbinol formation. The 9-HPOT and γ-9-HPOT were also specifically converted into the oxiranyl carbinols, 15,16- and 6,7-dehydro analogues of compound 1, respectively. The 13-HPOD was converted into erythro- and threo-isomers of oxiranyl carbinol, as well as oxiranyl vinyl carbinols. The obtained results allow assignment of the name "N. vectensis EAS" (NvEAS) to CYP443D1. The NvEAS is a first EAS detected in Cnidaria.

NMR structure, conformational dynamics, and biological activity of PsDef1 defensin from Pinus sylvestris.

Plants have developed a complex defense response system against pests and pathogens. Defensins, produced by plants as part of their innate immune response, form the family of small, basic, cysteine-rich proteins with activity primarily directed against fungal pathogens. In addition, plant defensins can show antibacterial activity and protease and insect amylase inhibitory activities. However, in gymnosperms, only antifungal activity of defensins has been described thus far. Here, we report antibacterial and insect α-amylase inhibition activities for defensin PsDef1 from P. sylvestris, the first defensin from gymnosperms with a broad range of biological activities described. We also report the solution NMR structure of PsDef1 and its dynamics properties assessed by a combination of experimental NMR and computational techniques. Collectively, our data provide an insight into structure, dynamics, and functional properties of PsDef1 that could be common between defensins from this taxonomic group.

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 and molecular cloning of CYP74Q1 gene: identification of Ranunculus acris leaf divinyl ether synthase.

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.

Oxylipin biosynthesis via an unprecedented 16-hydroperoxide lyase pathway in green tissues of cucumber (Cucumis sativus L.) plants.

The plant lipoxygenase cascade is a source of various regulatory oxylipins that play a role in cell signalling, stress adaptation, and immune response. Recently, we detected an unprecedented 16(S)-lipoxygenase, CsLOX3, in the leaves and fruit pericarp of cucumber (Cucumis sativus L.). In the present work, an array of products biosynthesized through the conversions of α-linolenic acid 16-hydroperoxide (16-HPOT) was detected. Firstly, a prominent 15-hydroxy-9,12-pentadecadienoic acid (Me/TMS) was detected, the product of hydroperoxide lyase (HPL) chain cleavage of 16-HPOT and further reduction of aldehyde 15-oxo-9,12-pentadecadienoic acid to alcohol. Besides, the presence of dicarboxylic acid, 3,6-pentadecadiene-1,15-dioic acid, was deduced from the detection of its catalytic hydrogenation product, pentadecane-1,15-dioic acid. Finally, 12,15-dihydroxypentadecanoic acid (Me/TMS) was detected amongst the hydrogenated products, thus indicating the presence of the parent 12,15-dihydroxy-9,13-pentadecadienoic acid. To confirm the proposed HPL chain cleavage, the 16(S)-HPOT was prepared and incubated with the recombinant cucumber HPL CYP74B6 enzyme. The CYP74B6 possessed high activity towards 16-HPOT. Chain cleavage yields the (9Z,12Z)-15-oxo-9,12-pentadecadienoic acid, undergoing a spontaneous isomerization into (9Z,13E)-15-oxo-9,13-pentadecadienoic acid. Thus, the cucumber plants as well as the recombinant cucumber HPL CYP74B6 possessed unprecedented 16-HPL activity, cleaving 16-HPOT into a C15 fragment, 15-oxo-9,12-pentadecadienoic acid, and a complementary volatile C3 fragment, propionic aldehyde. The 16-LOX/16-HPL route of oxylipin biosynthesis presents a novel facet of the plant LOX pathway.

The versatile CYP74 clan enzyme CYP440A19 from the European lancelet Branchiostoma lanceolatum biosynthesizes novel macrolactone, epoxydiene, and related oxylipins.

The present work reports the detection and cloning of a new CYP74 clan gene of the European lancelet (Branchiostoma lanceolatum) and the biochemical characterization of the recombinant protein CYP440A19. CYP440A19 possessed epoxyalcohol synthase (EAS) activity towards the 13-hydroperoxides of linoleic and α-linolenic acids, which were converted into oxiranylcarbinols, i.e., (11S,12R,13S)-11-hydroxy-12,13-epoxy derivatives. The conversion of 9-hydroperoxides produced distinct products. Linoleic acid 9(S)-hydroperoxide (9-HPOD) was mainly converted into 9,14-diol (10E,12E)-9,14-dihydroxy-10,12-octadecadienoic acid and macrolactone 9(S),10(R)-epoxy-11(E)-octadecen-13(S)-olide. In addition, (8Z)-colneleic acid was formed. Brief incubations of the enzyme with 9-HPOD in a biphasic system of hexane-water enabled the isolation of the short-lived 9,10-epoxydiene (9S,10R,11E,13E)-9,10-epoxy-11,13-octadecadienoic acid. The structure and stereochemistry of the epoxyalcohols, macrolactone, (8Z)-colneleic acid (Me), and 9,10-epoxydiene (Me) were confirmed by 1H-NMR, 1H-1H-COSY, 1H-13C-HSQC, and 1H-13C-HMBC spectroscopy. Macrolactone and cis-9,10-epoxydiene are novel products. The 9-hydroperoxide of α-linolenic acid was mainly converted into macrolactone 9(S),10(R)-epoxy-11(E),15(Z)-octadecadiene-13(S)-olide and a minority of divinyl ethers, particularly (8Z)-colnelenic acid. The versatility of enzyme catalysis, as well as the diversity of CYP74s and other enzymes involved in oxylipin biosynthesis, demonstrates the complexity of the lipoxygenase pathway in lancelets.

Green leaf divinyl ether synthase: gene detection, molecular cloning and identification of a unique CYP74B subfamily member.

Enzymes of the CYP74 family (P450 superfamily) play a key role in the plant lipoxygenase signalling cascade. Recently we detected a pathogen inducible divinyl ether synthase (DES) in flax leaves [Chechetkin, Blufard, Hamberg, Grechkin, 2008]. This prompted us to examine the CYP74 genes in the flax leaf transcriptome. Since the flax genome is not sequenced, we used the PCR approach with degenerate primers related to the conserved domains of selected CYP74 genes; this revealed several CYP74 transcripts in flax leaves. One transcript belongs to the previously described allene oxide synthase (LuAOS, CYP74A, GenBank ID: U00428.1). Another one contains the ORF (1473 bp) of an unknown CYP74B16 gene. Three more nearly identical sequences, including one expressed pseudogene, were also identified. The recombinant CYP74B16 protein expressed in Escherichia coli had 491 amino acid residues and MW of 56 kDa. The preferred substrate of this enzyme is the 13-hydroperoxide of α-linolenic acid, and the reaction product was identified by mass spectrometry, NMR and UV spectroscopy as the divinyl ether (9Z,11E)-12-[(1'Z,3'Z)-hexadienyloxy]-9,11-dodecadienoic acid, (ω5Z)-etherolenic acid. All previously known CYP74B subfamily enzymes are hydroperoxide lyases. The novel flax enzyme CYP74B16 (LuDES) is an unprecedented DES member of the CYP74B subfamily.

Detection of divinyl ether synthase CYP74H2 biosynthesizing (11Z)-etheroleic and (1'Z)-colnelenic acids in asparagus (Asparagus officinalis L.).

Divinyl ether synthases (DESs) are the enzymes occurring in numerous plant species and catalysing the dehydration of fatty acid hydroperoxides to divinyl ether oxylipins, playing self-defensive and antipathogenic roles in plants. Previously, the DES activities and divinyl ethers were detected in some monocotyledonous plants, including the asparagus (Asparagus officinalis L.). The cloning of the open reading frame of the CYP74H2 gene of asparagus and catalytic properties of the recombinant CYP74H2 protein are described in the present work. The CYP74H2 utilized the 13(S)-hydroperoxide of linoleic acid (13(S)-HPOD) as a preferred substrate and specifically converted it to the divinyl ether, (9Z,11Z)-12-[(1'E)-hexenyloxy]-9,11-dodecadienoic acid, (11Z)-etheroleic acid. The second most efficient substrate after the 13(S)-HPOD was the 9(S)-hydroperoxide of α-linolenic acid (9(S)-HPOT), which was converted to the previously undescribed product, (1'Z)-colnelenic acid. The 13(S)-hydroperoxide of α-linolenic acid (13(S)-HPOT) and 9(S)-hydroperoxide of linoleic acid (9(S)-HPOD) were less efficient substrates for CYP74H2. Both 13(S)-HPOT and 9(S)-HPOD were transformed to divinyl ethers, (11Z)-etherolenic and (1'Z)-colneleic acids, respectively. The CYP74H2 is a second cloned monocotyledonous DES after the garlic CYP74H1 and the first DES biosynthesizing the (1'Z)-colneleic and (1'Z)-colnelenic acids.

Lipoxygenase pathway in brown algae: The biosynthesis of novel oxylipins 'ectocarpins' by hydroperoxide bicyclase CYP5164A3 of Ectocarpus siliculosus.

The sequence encoding the CYP5164A3 of the brown alga Ectocarpus siliculosus (Stramenopiles, SAR) was heterologously expressed in E. coli cells. The resulting recombinant CYP74 clan-related protein CYP5164A3 possessed a selective activity towards the α-linolenic acid 13(S)-hydroperoxide (13-HPOTE) and eicosapentaenoic acid 15(S)-hydroperoxide (15-HPEPE). The major products were the heterobicyclic oxylipins. For instance, the 13-HPOTE was converted into plasmodiophorols A, B, and C formed at about 14:3:2 ratio. Plasmodiophorols A-C have been recently described as the products of enzyme hydroperoxide bicyclase CYP50918A1 of cercozoan Plasmodiophora brassicae (Rhizaria, SAR). Furthermore, an unknown compound 1 was detected. Purified product 1 (Me) was identified as a novel substituted 3-propenyl-6-oxabicyclo[3.1.0]hexane based on its MS and NMR spectral data. Conversion of 15-HPEPE by CYP5164A3 resulted in products 7 and 8, analogous to plasmodiophorols A and B. This work uncovered the CYP5164A3 as the first hydroperoxide bicyclase in brown algae. Apparently, this enzyme plays a crucial role in the biosynthesis of heterobicyclic oxylipins like hybridalactone, ecklonilactones, and related natural products, widespread in brown algae.