Lucia S. Mukhtarova

The lipoxygenase pathway in garlic (Allium sativum L.) bulbs" detection of the novel divinyl ether oxylipins

Incubations of [1‐14C]linoleic acid or [1‐14C]‐(9Z.11E, 13S)‐13‐hydropero xy‐9,11‐octadecadienoic acid (13‐HPOD) with juice of garlic bulbs lead to the formation of one predominant labelled product, viz., the novel divinyl ethery (9Z,11E, 1′E)‐12‐(1′‐hexenyloxy)‐9,11‐odecadienoic acid (‘etheroleic acid’). With lesser efficiency [1‐14C]α‐linolenic acid or [1‐14C](9Z,11E,13S,15Z)‐13‐hydroperoxy‐9,11,15‐octadecatrienoic acid (13‐HPOT) are converted in this way into (9Z,11E,1′E,3′Z)‐12‐(1′,3′‐hexadienyloxy)‐9,11‐dodecadienoic acid (‘etherolenic acid’). Thus, garlic bulbs possess the activity of a new 13‐hydroperoxide‐specific divinyl ether synthase.

Tomato CYP74C3 is a Multifunctional Enzyme not only Synthesizing Allene Oxide but also Catalyzing its Hydrolysis and Cyclization

The mechanism of the recombinant tomato allene oxide synthase (LeAOS3, CYP74C3) was studied. Incubations of linoleic acid (9S)‐hydroperoxide with dilute suspensions of LeAOS3 (10–20 s, 0 °C) yield mostly the expected allene oxide (12Z)‐9,10‐epoxy‐10,12‐octadecadienoic acid (9,10‐EOD), which was detected as its methanol‐trapping product. In contrast, the relative yield of 9,10‐EOD progressively decreased when the incubations were performed with fourfold, tenfold, or 80‐fold larger amounts of LeAOS3, while α‐ketol and the cyclopentenone rac‐cis‐10‐oxo‐11‐phytoenoic acid (10‐oxo‐PEA) became the predominant products. Both the α‐ketol and 10‐oxo‐PEA were also produced when LeAOS3 was exposed to preformed 9,10‐EOD, which was generated by maize allene oxide synthase (CYP74A). LeAOS3 also converted linoleic acid (13S)‐hydroperoxide into the corresponding allene oxide, but with about tenfold lower yield of cyclopentenone. The results indicate that in contrast to the ordinary allene oxide synthases (CYP74A subfamily), LeAOS3 (CYP74C subfamily) is a multifunctional enzyme, catalyzing not only the synthesis, but also the hydrolysis and cyclization of allene oxide.

Determinants governing the CYP74 catalysis: Conversion of allene oxide synthase into hydroperoxide lyase by site-directed mutagenesis

Bioinformatics analyses enabled us to identify the hypothetical determinants of catalysis by CYP74 family enzymes. To examine their recognition, two mutant forms F295I and S297A of tomato allene oxide synthase LeAOS3 (CYP74C3) were prepared by site‐directed mutagenesis. Both mutations dramatically altered the enzyme catalysis. Both mutant forms possessed the activity of hydroperoxide lyase, while the allene oxide synthase activity was either not detectable (F295I) or significantly reduced (S297A) compared to the wild‐type LeAOS3. Thus, both sites 295 and 297 localized within the “I‐helix central domain” (“oxygen binding domain”) are the primary determinants of CYP74 type of catalysis.

The lipoxygenase pathway in tulip (Tulipa gesneriana): detection of the ketol route

The in vitro metabolism of [1-(14)C]linoleate, [1-(14)C]linolenate and their 9(S)-hydroperoxides was studied in cell-free preparations from tulip (Tulipa gesneriana) bulbs, leaves and flowers. Linoleate and its 9-hydroperoxide were converted by bulb and leaf preparations into three ketols: (12Z)-9-hydroxy-10-oxo-12-octadecadienoic acid (alpha-ketol), (11E)-10-oxo-13-hydroxy-11-octadecadienoic acid (gamma-ketol) and a novel compound, (12Z)-10-oxo-11-hydroxy-12-octadecadienoic acid (10,11-ketol), in the approximate molar proportions of 10:3:1. The corresponding 15, 16-dehydro alpha- and gamma-ketols were the main metabolites of [1-(14)C]linolenate and its 9-hydroperoxide. Thus bulbs and leaves possessed 9-lipoxygenase and allene oxide synthase activities. Incubations with flower preparations gave alpha-ketol hydro(pero)xides as predominant metabolites. Bulb and leaf preparations possessed a novel enzyme activity, gamma-ketol reductase, which reduces gamma-ketol to 10-oxo-13-hydroxyoctadecanoic acid (dihydro-gamma-ketol) in the presence of NADH. Exogenous linolenate 13(S)-hydroperoxide was converted mostly into chiral (9S,13S)-12-oxo-10-phytodienoate (99.5% optical purity) by bulb preparations, while [1-(14)C]linolenate was a precursor for ketols only. Thus tulip bulbs possess abundant allene oxide cyclase activity, the substrate for which is linolenate 13(S)-hydroperoxide, even though 13(S)-lipoxygenase products were not detectable in the bulbs. The majority of the cyclase activity was found in the microsomes (10(5) g pellet). Cyclase activity was not found in the other tissues examined, but only in the bulbs. The ketol route of the lipoxygenase pathway, mediated by 9-lipoxygenase and allene oxide synthase activities, has not been detected previously in the vegetative organs of any plant species.

Hydroperoxide lyase cascade in pea seedlings: Non-volatile oxylipins and their age and stress dependent alterations.

The profiles of non-volatile oxylipins of pea (Pisum sativum) seedlings were examined by gas chromatography-mass spectrometry after invitro incubation with α-linolenic acid. The 13-lipoxygenase/hydroperoxide lyase (HPL) products were predominant in the leaves, while the roots possess both 13- and 9-HPL products. Allene oxide synthase (AOS) and divinyl ether synthase (DES) products were not detected in the leaves or in the roots of any age. The HPL cascade produces a diversity of oxylipins, including the compounds (2E)-4-hydroxy-traumatic, (10E)-9,12-dihydroxy-10-dodecenoic and 9,12-dihydroxydodecanoic acids, as well as (2E)-4-hydroxy-2-nonenoic acid, which has not yet been detected in plants. Oxylipin patterns were altered by infection, water deficit, as well as by plant age. Infection caused the specific strong accumulation of azelaic (nonane-1,9-dioic) acid in the leaves. The azelaic acid content in the aged (14 and 18day-old) leaves was significantly higher than in the younger leaves. Water deficit induced the accumulation of (2E)-4-hydroxy-2-nonenoic acid and (2E)-traumatic acid in the roots. Results demonstrate that: (1) the HPL cascade is the predominant branch of the lipoxygenase pathway in pea seedlings; (2) the HPL products may have the regulatory role both in growth control and adaptation.

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.

Detection of a pathway from linoleate to a novel cyclopentenone: cis-12-oxo-10-phytoenoic acid in sunflower roots.

The lipoxygenase pathway in sunflower roots was studied in vitro. A preliminary incubation of linoleic acid with 15 000 g supernatant of homogenate of sunflower roots (1.5–6 days after germination) revealed the predominant activity of 13‐lipoxygenase. The exogenously added linoleic acid 13‐hydroperoxide is further utilized through two competing pathways. One of them is directed towards formation of the ketodiene (9Z,11E)‐13‐oxooctadeca‐9,11‐dienoic acid. The second pathway, which is controlled by allene oxide synthase, leads to the formation of an α‐ketol and a novel cyclopentenone, rac‐cis‐12‐oxo‐10‐phytoenoic acid (12‐oxo‐PEA) via a short‐lived allene oxide. Unexpectedly, the cyclopentenone 12‐oxo‐PEA is the predominant allene oxide synthase product. Identification of cis‐12‐oxo‐PEA was confirmed by its UV, mass, 1H NMR and 2D‐COSY spectral data. The highest yield of 12‐oxo‐PEA is observed in very young roots (1.5–2 days after germination). The results of methanol‐trapping experiments demonstrate that both 12‐oxo‐PEA and α‐ketol are formed through the unstable allene oxide intermediate, (9Z)‐12,13‐epoxyoctadeca‐9,11‐dienoic acid, which is the primary product of allene oxide synthase. Since 12‐oxo‐PEA is a jasmonate congener, its biosynthesis in plants might be of physiological importance.

Detection of an enol intermediate in the hydroperoxide lyase chain cleavage reaction

Guava (Psidium guajava) hydroperoxide lyase (HPL) preparations were incubated with [1‐14C](9Z,11E,13S,15Z)‐13‐hydroperoxy‐9,11,15‐octadecatrienoic acid for 1 min at 0°C, followed by rapid extraction/trimethylsilylation. Analysis of the trimethylsilylated products by gas chromatography–mass spectrometry and radio‐high‐performance liquid chromatography revealed a single predominant 14C‐labelled compound, identified by its 1H‐nuclear magnetic resonance, ultraviolet and mass spectra as the trimethylsilyl ether/ester of (9Z,11E)‐12‐hydroxy‐9,11‐dodecadienoic acid. Longer time incubations afford smaller yield of this enol due to its partial tautomerization into (9Z)‐12‐oxo‐9‐dodecenoic acid. The data obtained demonstrate that formation of (9Z)‐12‐oxo‐9‐dodecenoic acid in the HPL reaction is preceded by unstable enol oxylipin, and further suggest that hemiacetals are the true products of HPL catalysis.

The keto-enol tautomerism and the redox conversions of α-ketol fatty acids

Abstract The main conversions of α-ketol fatty acids (18:2 and 18:3), accompanying their redox interactions with 2,6-dichlorophenolindophenol (DCPIP) and copper(II) acetate, were studied. The highest rates of oxidation with DCPIP were observed in the alkaline water solutions. In the acidic aqueous media or in organic solvents, reaction with DCPIP proceeded much more slowly. The presence of oxygen significantly altered the direction but not the kinetics of reaction. The predominant products of aerobic reaction with DCPIP under alkaline conditions were α-ketol hydroperoxides. Similar incubations under argon atmosphere, excluding hydroperoxide formation, afforded 9,13-dihydroxy-12-oxo and 9,12-dioxo-13-hydroxy acids. The latter was also the main product of reaction with copper acetate, along with 9-acetoxy-12-oxo-13-hydroxy acid. The free radical mechanism of the observed conversions is proposed.

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.