12-Oxo-10-glutathionyl-5,8,14-eicosatrienoic acid (TOG10), a novel glutathione-containing eicosanoid generated via the 12-lipoxygenase pathway in human platelets.

Biologically active glutathione (GSH) conjugates of oxygenated fatty acids comprise a group of pro- and anti-inflammatory lipid mediators. While arachidonic acid (AA)-derived conjugates, as the cysteinyl leukotrienes (cys-LTs) and eoxins (EXs) have pro-inflammatory properties, conjugates in tissue regeneration (CTRs) biosynthesized from docosahexaenoic acid (DHA) exhibit pro-resolving activity. Human platelets express abundant amounts of platelet-type 12-lipoxygenase (pt12-LOX) and leukotriene C4 synthase (LTC4S). However, the only two described GSH conjugates formed by platelets are the AA-derived cys-LTs and the recently reported maresin CTRs (MCTRs). While cys-LTs are biosynthesized in a transcellular mechanism via the action of 5-LOX and LTC4S, MCTR1 is formed by 12-LOX and a yet unidentified GSH S-transferase (GST). Here, we present a novel GSH conjugate formed from AA via the 12-LOX pathway in human platelets. The 12-oxo-glutathione adduct, 12-oxo-10-glutathionyl-5,8,14-eicosatrienoic acid (TOG10), was identified by mass spectrometry using positive electrospray ionization. The structural proposal is supported by fragmentation data of the labeled metabolite obtained after incubation of deuterated AA (AA-d8). In platelets as well as in HEK293 cells stably expressing pt12-LOX, TOG10 biosynthesis was inhibited by the 12-LOX inhibitor ML-355 (5 µM), which confirms the involvement of pt12-LOX. Interestingly, TOG10 was formed independently of LTC4S in platelets. This is in accordance with the observation that the conjugate was also generated by AA-stimulated HEK_12-LOX cells in absence of LTC4S. Nevertheless, TOG10 can also be formed by LTC4S as the biosynthesis in HEK_12-LOX_LTC4S cells was reduced by the specific LTC4S inhibitor TK04a. In summary, TOG10 was identified as a new AA-derived GSH conjugate generated in human platelets via the action of pt12-LOX in combination with a GST.

Mammalian-Like Inflammatory and Pro-Resolving Oxylipins in Marine Algae.

Oxylipins constitute a family of oxidized fatty acids, that are well known as tissue hormones in mammals. They contribute to inflammation and its resolution. The major classes of these lipid mediators are inflammatory prostaglandins (PGs) and leukotrienes (LTs) as well as pro-resolving resolvins (Rvs). Understanding their biosynthetic pathways and modes of action is important for anti-inflammatory interventions. Besides mammals, marine algae also biosynthesize mammalian-like oxylipins and thus offer new opportunities for oxylipin research. They provide prolific sources for these compounds and offer unique opportunities to study alternative biosynthetic pathways to the well-known lipid mediators. Herein, we discuss recent findings on the biosynthesis of oxylipins in mammals and algae including an alternative pathway to prostaglandin E2 , a novel pathway to a precursor of leukotriene B4 , and the production of resolvins in algae. We evaluate the pharmacological potential of the algal metabolites with implications in health and disease.

15-Hydroperoxy-PGE2 : Intermediate in Mammalian and Algal Prostaglandin Biosynthesis.

Arachidonic-acid-derived prostaglandins (PGs), specifically PGE2 , play a central role in inflammation and numerous immunological reactions. The enzymes of PGE2 biosynthesis are important pharmacological targets for anti-inflammatory drugs. Besides mammals, certain edible marine algae possess a comprehensive repertoire of bioactive arachidonic-acid-derived oxylipins including PGs that may account for food poisoning. Described here is the analysis of PGE2 biosynthesis in the red macroalga Gracilaria vermiculophylla that led to the identification of 15-hydroperoxy-PGE2 , a novel precursor of PGE2 and 15-keto-PGE2 . Interestingly, this novel precursor is also produced in human macrophages where it represents a key metabolite in an alternative biosynthetic PGE2 pathway in addition to the well-established arachidonic acid-PGG2 -PGH2 -PGE2 route. This alternative pathway of mammalian PGE2 biosynthesis may open novel opportunities to intervene with inflammation-related diseases.

A Fivefold Parallelized Biosynthetic Process Secures Chlorination of Armillaria mellea (Honey Mushroom) Toxins.

The basidiomycetous tree pathogen Armillaria mellea (honey mushroom) produces a large variety of structurally related antibiotically active and phytotoxic natural products, referred to as the melleolides. During their biosynthesis, some members of the melleolide family of compounds undergo monochlorination of the aromatic moiety, whose biochemical and genetic basis was not known previously. This first study on basidiomycete halogenases presents the biochemical in vitro characterization of five flavin-dependent A. mellea enzymes (ArmH1 to ArmH5) that were heterologously produced in Escherichia coli. We demonstrate that all five enzymes transfer a single chlorine atom to the melleolide backbone. A 5-fold, secured biosynthetic step during natural product assembly is unprecedented. Typically, flavin-dependent halogenases are categorized into enzymes acting on free compounds as opposed to those requiring a carrier-protein-bound acceptor substrate. The enzymes characterized in this study clearly turned over free substrates. Phylogenetic clades of halogenases suggest that all fungal enzymes share an ancestor and reflect a clear divergence between ascomycetes and basidiomycetes.

14,17,18-Trihydroxy-Eicosatetraenoic Acid: A Novel Pro-Resolving Lipid Mediator from Marine Microalgae.

Specialized pro-resolving mediators (SPMs) are enzymatically oxygenated derivatives of polyunsaturated fatty acids that function as central immunoregulators in mammals. Among them are resolvins (Rvs) that stimulate the clearance of harmful stimuli and limit pro-inflammatory processes. Because of their beneficial features and their high potency, SPMs are promising molecules for anti-inflammatory therapy. Besides mammals, also marine algae form lipid mediators such as prostaglandins and leukotrienes. In particular, microalgae are attractive candidates for the production of bioactive high-value metabolites. Here, we identified the diatom Cylindrotheca closterium as a prolific producer of SPMs. The diatom forms RvE3 and novel structurally related eicosanoids, including 14S/R,17R,18R-trihydroxy-eicosatetraenoic acid, which displays inflammation-resolving and anti-inflammatory bioactivities. This pro-resolving compound might enable advancements in anti-inflammatory therapy in mammals.

An Alternative Pathway to Leukotriene B4 Enantiomers Involving a 1,8-Diol-Forming Reaction of an Algal Oxylipin.

Oxidized lipids function as tissue hormones in mammals. An important class of these oxylipins are the immunomodulatory leukotrienes (LTs). Besides mammals, marine algae produce bioactive oxylipins, including LTs. The novel acid-labile oxylipin, (5 R,8 S)-dihydroxy eicosatetraenoic acid, from the edible alga Gracilaria vermiculophylla rearranges via a 1,8-diol-forming mechanism to inflammatory LTB4 enantiomers that exhibit an enantio-specific potency toward LTB4 receptor 1. This alternative pathway to a well-known leukotriene may explain food poisoning after Gracilaria consumption.