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.

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

The genome of the neotropical fruit bat was recently sequenced, revealing an unexpected gene encoding a plant-like protein, CYP74C44, which shares ca. 90% sequence identity with the putative CYP74C of . The preparation and properties of the recombinant CYP74C44 are described in the present work.

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).

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.).

Gene Expression Analysis of Potato ( L.) Lipoxygenase Cascade and Oxylipin Signature under Abiotic Stress.

The metabolism of polyunsaturated fatty acids through the lipoxygenase-catalyzed step and subsequent reactions is referred to as the lipoxygenase (LOX) pathway. The components of this system, such as jasmonates, are involved in growth, development and defense reactions of plants. In this report, we focus on dynamics of expression of different LOX pathway genes and activities of target enzymes with three abiotic stress factors: darkness, salinity and herbicide toxicity.

Oxylipin biosynthesis in spikemoss Selaginella moellendorffii: Identification of allene oxide synthase (CYP74L2) and hydroperoxide lyase (CYP74L1).

Nonclassical P450s of the CYP74 family catalyse the secondary conversions of fatty acid hydroperoxides to bioactive oxylipins in plants. The model organism, spikemoss Selaginella moellendorffii Hieron, possesses at least ten CYP74 genes of novel J, K, L, and M subfamilies. The cloning of three CYP74L genes and catalytic properties of recombinant proteins are described in the present work.

Detection of the First Epoxyalcohol Synthase/Allene Oxide Synthase (CYP74 Clan) in the Lancelet (, 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 (, Chordata) was biochemically characterized in the present work.

Lipoxygenase pathway in model bryophytes: 12-oxo-9(13),15-phytodienoic acid is a predominant oxylipin in Physcomitrella patens.

The model moss Physcomitrella patens and liverwort Marchantia polymorpha possess all enzymatic machinery responsible for the initial stages of jasmonate pathway, including the active 13(S)-lipoxygenase, allene oxide synthase (AOS) and allene oxide cyclase (AOC). At the same time, the jasmonic acid is missing from both P. patens and M.

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.

Epoxyalcohol synthase activity of the CYP74B enzymes of higher plants.

The CYP74B subfamily of fatty acid hydroperoxide transforming cytochromes P450 includes the most common plant enzymes. All CYP74Bs studied yet except the CYP74B16 (flax divinyl ether synthase, LuDES) and the CYP74B33 (carrot allene oxide synthase, DcAOS) are 13-hydroperoxide lyases (HPLs, synonym: hemiacetal synthases). The results of present work demonstrate that additional products (except the HPL products) of fatty acid hydroperoxides conversion by the recombinant StHPL (CYP74B3, Solanum tuberosum), MsHPL (CYP74B4v1, Medicago sativa), and CsHPL (CYP74B6, Cucumis sativus) are epoxyalcohols.

Catalysis by allene oxide synthases (CYP74A and CYP74C): Alterations by the Phe/Leu mutation at the SRS-1 region.

The CYP74 family of cytochromes P450 includes four fatty acid hydroperoxide metabolizing enzymes: allene oxide synthase (AOS), hydroperoxide lyase (HPL), divinyl ether synthase, and epoxyalcohol synthase (EAS). All P450s have six substrate recognition sites (SRSs) in their structures. Some CYP74 mutations in SRSs leading to their interconversions including substitutions in "F/L toggle" (SRS-1 region) were reported before.

Plant hydroperoxide-cleaving enzymes (CYP74 family) function as hemiacetal synthases: Structural proof of hemiacetals by NMR spectroscopy.

Hydroperoxide lyases (HPLs) of the CYP74 family (P450 superfamily) are widely distributed enzymes in higher plants and are responsible for the stress-initiated accumulation of short-chain aldehydes. Fatty acid hydroperoxides serve as substrates for HPLs; however, details of the HPL-promoted conversion are still incompletely understood. In the present work, we report first time the micropreparative isolation and the NMR structural studies of fatty acid hemiacetal (TMS/TMS), the short-lived HPL product.

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).

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.

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).

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.

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.

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.

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.

Thermal conversions of fatty acid peroxides to cyclopentenones: a biomimetic model for allene oxide synthase pathway.

The trimethylsilyl (TMS) peroxides of linoleic acid 9(S)-hydroperoxide (TMS or Me esters) were subjected to gas chromatography-mass spectrometry (GC-MS) analyses. The cyclopentenones, trans- and cis-10-oxo-11-phytoenoic acid (10-oxo-PEA, Me or TMS esters) were first time detected as the products of TMS-peroxide thermal conversions. The major products were ketodienes, epoxyalcohols, hemiacetals and decadienals.

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.

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.

Detection of divinyl ether synthase in Lily-of-the-Valley (Convallaria majalis) roots.

Incubations of linoleic acid with cell-free preparations from Lily-of-the-Valley (Convallaria majalis L., Ruscaceae) roots revealed the presence of 13-lipoxygenase and divinyl ether synthase (DES) activities. Exogenous linoleic acid was metabolized predominantly into (9Z,11E,1'E)-12-(1'-hexenyloxy)-9,11-dodecadienoic (etheroleic) acid.

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.

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 degrees 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 alpha-ketol and the cyclopentenone rac-cis-10-oxo-11-phytoenoic acid (10-oxo-PEA) became the predominant products.