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.

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.

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 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.

The CYP74B and CYP74D divinyl ether synthases possess a side hydroperoxide lyase and epoxyalcohol synthase activities that are enhanced by the site-directed mutagenesis.

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.