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.

Lipoxygenase functions in 1O2 production during root responses to osmotic stress

Drought induces osmotic stress in roots, a condition simulated by the application of high-molecular-weight polyethylene glycol. Osmotic stress results in the reduction of Arabidopsis thaliana root growth and production of 1O2 from an unknown non-photosynthetic source. Reduced root growth can be alleviated by application of the 1O2 scavenger histidine. Here we examined the possibility that 1O2 production involves Russell reactions occurring among the enzymatic products of lipoxygenases, the fatty acid hydroperoxides. Lipoxygenase (LOX) activity was measured for purified soybean (Glycine max) LOX1 and in crude Arabidopsis root extracts using linoleic acid as substrate. Formation of the 13(S)-Hydroperoxy-9(Z),11(E)-octadecadienoic acid product was inhibited by salicylhdroxamic acid, which is a lipoxygenase inhibitor, but not by histidine, whereas 1O2 production was inhibited by both. D2O, which specifically extends the half-life of 1O2, augmented the lipoxygenase-dependent generation of 1O2, as expected from a Russell-type reaction. The addition of linoleic acid to roots stimulated 1O2 production and inhibited growth, suggesting that the availability of lipoxygenase substrate is a rate-limiting step. Indeed, water stress rapidly increased linoleic and linolenic acids by 2.5-fold in roots. Mutants with root-specific microRNA repression of lipoxygenases showed downregulation of lipoxygenase protein and activity. The lines with downregulated lipoxygenase displayed significantly less 1O2 formation, improved root growth in osmotic stress, and an altered transcriptome response compared to wild type. The results show that lipoxygenases can serve as an enzymatic source of ‘dark’ 1O2 during osmotic stress and demonstrate a role for 1O2 in defining the physiological response.

COMPARATIVE ANALYSIS OF HEMOPROTEINS (CYTOCHROME P450 AND CATA-LASE) PARTICIPATING IN THE BIOSYNTHESIS OF OXYLIPINS IN PLANTS AND ANIMALS

The article discusses the comparative characteristics of hemoproteins involved in the metabolism of fatty acid hydroperoxides in plants and corals.

COMPARATIVE ANALYSIS OF HEMOPROTEINS (CYTOCHROME P450 AND CATA-LASE) PARTICIPATING IN THE BIOSYNTHESIS OF OXYLIPINS IN PLANTS AND ANIMALS

The article discusses the comparative characteristics of hemoproteins involved in the metabo-lism of fatty acid hydroperoxides in plants and corals.

Isolation of lactic acid bacteria capable of reducing environmental alkyl and fatty acid hydroperoxides, and the effect of their oral administration on oxidative-stressed nematodes and rats

Reinforcement of hydroperoxide-eliminating activity in the intestines and colon should prevent associated diseases. We previously isolated a lactic acid bacterium,Pediococcus pentosaceusBe1, that facilitates a 2-electron reduction of hydrogen peroxide to water. In this study, we successfully isolated an alternative lactic acid bacterium,Lactobacillus plantarumP1-2, that can efficiently reduce environmental alkyl hydroperoxides and fatty acid hydroperoxides to their corresponding hydroxy derivatives through a 2-electron reduction. Each strain exhibited a wide concentration range with regard to the environmental reducing activity for each hydroperoxide. Given this, the two lactic acid bacteria were orally administered to the oxygen-sensitive short-lived nematode mutant, and this resulted in a significant expansion of its lifespan. This observation suggests thatP. pentosaceusBe1 andL. plantarumP1-2 inhibit internal oxidative stress. To determine the specific organs involved in this response, we performed a similar experiment in rats, involving induced lipid peroxidation by iron-overloading. We observed that onlyL. plantarumP1-2 inhibited colonic mucosa lipid peroxidation in rats with induced oxidative stress.

Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite

Organic hydroperoxide resistance (Ohr) enzymes are unique Cys-based, lipoyl-dependent peroxidases. Here, we investigated the involvement of Ohr in bacterial responses toward distinct hydroperoxides. In silico results indicated that fatty acid (but not cholesterol) hydroperoxides docked well into the active site of Ohr fromXylella fastidiosaand were efficiently reduced by the recombinant enzyme as assessed by a lipoamide-lipoamide dehydrogenase–coupled assay. Indeed, the rate constants between Ohr and several fatty acid hydroperoxides were in the 107–108M−1⋅s−1range as determined by a competition assay developed here. Reduction of peroxynitrite by Ohr was also determined to be in the order of 107M−1⋅s−1at pH 7.4 through two independent competition assays. A similar trend was observed when studying the sensitivities of a ∆ohrmutant ofPseudomonas aeruginosatoward different hydroperoxides. Fatty acid hydroperoxides, which are readily solubilized by bacterial surfactants, killed the ∆ohrstrain most efficiently. In contrast, both wild-type and mutant strains deficient for peroxiredoxins and glutathione peroxidases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because the ∆ohrmutant was more sensitive than wild type to 3-morpholinosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In the case of H2O2insult, cells treated with 3-amino-1,2,4-triazole (a catalase inhibitor) were the most sensitive. Furthermore, fatty acid hydroperoxide and SIN-1 both induced Ohr expression in the wild-type strain. In conclusion, Ohr plays a central role in modulating the levels of fatty acid hydroperoxides and peroxynitrite, both of which are involved in host–pathogen interactions.