Heme-catalyzed degradation of linoleate 9-hydroperoxide (9-HPODE) forms two allylic epoxy-ketones via a proposed pseudo-symmetrical diepoxy radical intermediate.

Heme-initiated decomposition of unsaturated fatty acid hydroperoxides creates alkoxyl radicals that propagate a complex series of reactions to hydroxy, keto, epoxy and aldehydic products. Herein, among the products from the hematin-catalyzed degradation of 9-hydroperoxy-linoleic acid (9-HPODE), we observed a double peak on normal-phase HPLC that resolved on RP-HPLC into equal proportions of two epoxy-allylic ketones with identical UV spectra. Their proton NMR spectra were also indistinguishable and consistent with 9,10--epoxy-11-13-keto- and 9-keto-10-12,13--epoxy-octadecenoic acids.

The Michael addition of thiols to 13-oxo-octadecadienoate (13-oxo-ODE) with implications for LC-MS analysis of glutathione conjugation.

Unsaturated fatty acid ketones with αβ,γδ conjugation are susceptible to Michael addition of thiols, with unresolved issues on the site of adduction and precise structures of the conjugates. Herein we reacted 13-keto-octadecadienoic acid (13-oxo-ODE or 13-KODE) with glutathione (GSH), N-acetyl-cysteine, and β-mercaptoethanol and identified the adducts. HPLC-UV analyses indicated none of the products exhibit a conjugated enone UV chromophore, a result that conflicts with the literature and is relevant to the mass spectral interpretation of 1,4 versus 1,6 thiol adduction.

Fatty acids of lipoproteins CagT and Lpp20.

Bacterial lipoproteins are post-translationally modified by the addition of acyl chains that anchor the protein to bacterial membranes. This modification includes two ester-linked and one amide-linked acyl chain on lipoproteins from Gram-negative bacteria. lipoproteins have important functions in pathogenesis (including delivering the CagA oncoprotein to mammalian cells) and are recognized by host innate and adaptive immune systems.

Evaluation of ω-alkynyl-labeled linoleic and arachidonic acids as substrates for recombinant lipoxygenase pathway enzymes.

ω-Alkynyl-fatty acids can be used as probes for covalent binding to intracellular macromolecules. To inform future in vivo studies, we determined the rates of reaction of ω-alkynyl-labeled linoleate with recombinant enzymes of the skin 12R-lipoxygenase (12R-LOX) pathway involved in epidermal barrier formation (12R-LOX, epidermal lipoxygenase-3 (eLOX3), and SDR9C7). We also examined the reactivity of ω-alkynyl-arachidonic acid with representative lipoxygenase enzymes employing either "carboxyl end-first" substrate binding (5S-LOX) or "tail-first" (platelet-type 12S-LOX).

Recombinant PNPLA1 catalyzes the synthesis of acylceramides and acyl acids with selective incorporation of linoleic acid.

Loss-of-function mutations in patatin-like phospholipase domain-containing protein 1 (PNPLA1) cause autosomal recessive congenital ichthyosis, and altered PNPLA1 activity is implicated in the pathogenesis of atopic dermatitis and other common skin diseases. To examine the hypothesis that PNPLA1 catalyzes the synthesis of acylceramides and acyl acids, we expressed and partially purified a soluble, truncated form of PNPLA1 in Escherichia coli, (PNPLA1) along with the related protein PNPLA2 (ATGL, adipose triglyceride lipase) and coactivator CGI-58. Liposomal substrates were incubated with recombinant enzymes for 0.

Two C18 hydroxy-cyclohexenone fatty acids from mammalian epidermis: Potential relation to 12R-lipoxygenase and covalent binding of ceramides.

A key requirement in forming the water permeability barrier in the mammalian epidermis is the oxidation of linoleate esterified in a skin-specific acylceramide by the sequential actions of 12R-lipoxygenase, epidermal lipoxygenase-3, and the epoxyalcohol dehydrogenase SDR9C7 (short-chain dehydrogenase-reductase family 7 member 9). By mechanisms that remain unclear, this oxidation pathway promotes the covalent binding of ceramides to protein, forming a critical structure of the epidermal barrier, the corneocyte lipid envelope. Here, we detected, in porcine, mouse, and human epidermis, two novel fatty acid derivatives formed by KOH treatment from precursors covalently bound to protein: a "polar" lipid chromatographing on normal-phase HPLC just before omega-hydroxy ceramide and a "less polar" lipid nearer the solvent front.

Dehydrogenase reductase 9 (SDR9C4) and related homologs recognize a broad spectrum of lipid mediator oxylipins as substrates.

Bioactive oxylipins play multiple roles during inflammation and in the immune response, with termination of their actions partly dependent on the activity of yet-to-be characterized dehydrogenases. Here, we report that human microsomal dehydrogenase reductase 9 (DHRS9, also known as SDR9C4 of the short-chain dehydrogenase/reductase (SDR) superfamily) exhibits a robust oxidative activity toward oxylipins with hydroxyl groups located at carbons C9 and C13 of octadecanoids, C12 and C15 carbons of eicosanoids, and C14 carbon of docosanoids. DHRS9/SDR9C4 is also active toward lipid inflammatory mediator dihydroxylated Leukotriene B and proresolving mediators such as tri-hydroxylated Resolvin D1 and Lipoxin A, although notably, with lack of activity on the 15-hydroxyl of prostaglandins.

Monolayer autoxidation of arachidonic acid to epoxyeicosatrienoic acids as a model of their potential formation in cell membranes.

In light of the importance of epoxyeicosatrienoic acids (EETs) in mammalian pathophysiology, a nonenzymatic route that might form these monoepoxides in cells is of significant interest. In the late 1970s, a simple system of arranging linoleic acid molecules on a monolayer on silica was devised and shown to yield monoepoxides as the main autoxidation products. Here, we investigated this system with arachidonic acid and characterized the primary products.

Lipidomic and transcriptional analysis of the linoleoyl-omega-hydroxyceramide biosynthetic pathway in human psoriatic lesions.

A complex assembly of lipids including fatty acids, cholesterol, and ceramides is vital to the integrity of the mammalian epidermal barrier. The formation of this barrier requires oxidation of the substrate fatty acid, linoleic acid (LA), which is initiated by the enzyme 12R-lipoxygenase (LOX). In the epidermis, unoxidized LA is primarily found in long-chain acylceramides termed esterified omega-hydroxy sphingosine (EOS)/phytosphingosine/hydroxysphingosine (collectively EOx).

Analysis of 12/15-lipoxygenase metabolism of EPA and DHA with special attention to authentication of docosatrienes.

A proposed beneficial impact of highly unsaturated "fish oil" fatty acids is their conversion by lipoxygenase (LOX) enzymes to specialized proresolving lipid mediators, including 12/15-LOX products from EPA and DHA. The transformations of DHA include formation of docosatrienes, named for the distinctive conjugated triene of the double bonds. To further the understanding of biosynthetic pathways and mechanisms, herein we meld together biosynthesis and NMR characterization of the unstable leukotriene A (LTA)-related epoxide intermediates formed by recombinant human 15-LOX-1, along with identification of the stable enzymatic products, and extend the findings into the 12/15-LOX metabolism in resident murine peritoneal macrophages.

Epoxide hydrolase 3 (Ephx3) gene disruption reduces ceramide linoleate epoxide hydrolysis and impairs skin barrier function.

The mammalian epoxide hydrolase (EPHX)3 is known from in vitro experiments to efficiently hydrolyze the linoleate epoxides 9,10-epoxyoctadecamonoenoic acid (EpOME) and epoxyalcohol 9R,10R-trans-epoxy-11E-13R-hydroxy-octadecenoate to corresponding diols and triols, respectively. Herein we examined the physiological relevance of EPHX3 to hydrolysis of both substrates in vivo. Ephx3 mice show no deficiency in EpOME-derived plasma diols, discounting a role for EPHX3 in their formation, whereas epoxyalcohol-derived triols esterified in acylceramides of the epidermal 12R-lipoxygenase pathway are reduced.

SDR9C7 catalyzes critical dehydrogenation of acylceramides for skin barrier formation.

The corneocyte lipid envelope, composed of covalently bound ceramides and fatty acids, is important to the integrity of the permeability barrier in the stratum corneum, and its absence is a prime structural defect in various skin diseases associated with defective skin barrier function. SDR9C7 encodes a short-chain dehydrogenase/reductase family 9C member 7 (SDR9C7) recently found mutated in ichthyosis. In a patient with SDR9C7 mutation and a mouse Sdr9c7-KO model, we show loss of covalent binding of epidermal ceramides to protein, a structural fault in the barrier.

Revising the structure of a new eicosanoid from human platelets to 8,9-11,12-diepoxy-13-hydroxyeicosadienoic acid.

Eicosanoids are critical mediators of fever, pain, and inflammation generated by immune and tissue cells. We recently described a new bioactive eicosanoid generated by cyclooxygenase-1 (COX-1) turnover during platelet activation that can stimulate human neutrophil integrin expression. On the basis of mass spectrometry (MS/MS and MS), stable isotope labeling, and GC-MS analysis, we previously proposed a structure of 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (DXA).

An ensemble of lipoxygenase structures reveals novel conformations of the Fe coordination sphere.

The regio- and stereo-specific oxygenation of polyunsaturated fatty acids is catalyzed by lipoxygenases (LOX); both Fe and Mn forms of the enzyme have been described. Structural elements of the Fe and Mn coordination spheres and the helical catalytic domain in which the metal center resides are highly conserved. However, animal, plant, and microbial LOX each have distinct features.

Residual cyclooxygenase activity of aspirin-acetylated COX-2 forms 15 R-prostaglandins that inhibit platelet aggregation.

Aspirin (acetylsalicylic acid) inhibits prostaglandin (PG) synthesis by transfer of its acetyl group to a serine residue in the cyclooxygenase (COX) active site. Acetylation of Ser530 inhibits catalysis by preventing access of arachidonic acid substrate in the COX-1 isoenzyme. Acetylated COX-2, in contrast, gains a new catalytic activity and forms 15 R hydroxy-eicosatetraenoic acid (15 R-HETE) as alternate product.

Thiol Reactivity of Curcumin and Its Oxidation Products.

The polypharmacological effects of the turmeric compound curcumin may be partly mediated by covalent adduction to cellular protein. Covalent binding to small molecule and protein thiols is thought to occur through a Michael-type addition at the enone moiety of the heptadienedione chain connecting the two methoxyphenol rings of curcumin. Here we show that curcumin forms the predicted thiol-Michael adducts with three model thiols, glutathione, N-acetylcysteine, and β-mercaptoethanol.

Catalytic activities of mammalian epoxide hydrolases with and fatty acid epoxides relevant to skin barrier function.

Lipoxygenase (LOX)-catalyzed oxidation of the essential fatty acid, linoleate, represents a vital step in construction of the mammalian epidermal permeability barrier. Analysis of epidermal lipids indicates that linoleate is converted to a trihydroxy derivative by hydrolysis of an epoxy-hydroxy precursor. We evaluated different epoxide hydrolase (EH) enzymes in the hydrolysis of skin-relevant fatty acid epoxides and compared the products to those of acid-catalyzed hydrolysis.

Oxidation of C18 Hydroxy-Polyunsaturated Fatty Acids to Epoxide or Ketone by Catalase-Related Hemoproteins Activated with Iodosylbenzene.

Small catalase-related hemoproteins with a facility to react with fatty acid hydroperoxides were examined for their potential mono-oxygenase activity when activated using iodosylbenzene. The proteins tested were a Fusarium graminearum 41 kD catalase hemoprotein (Fg-cat, gene FGSG_02217), a Pseudomonas fluorescens Pfl01 catalase (37.5 kD, accession number WP_011333788.

A fungal catalase reacts selectively with the 13S fatty acid hydroperoxide products of the adjacent lipoxygenase gene and exhibits 13S-hydroperoxide-dependent peroxidase activity.

The genome of the fungal plant pathogen Fusarium graminearum harbors six catalases, one of which has the sequence characteristics of a fatty acid peroxide-metabolizing catalase. We cloned and expressed this hemoprotein (designated as Fg-cat) along with its immediate neighbor, a 13S-lipoxygenase (cf. Brodhun et al.

Biomimetic synthesis of hemiketal eicosanoids for biological testing.

The hemiketal (HK) eicosanoids HKE and HKD are the major products resulting from the biosynthetic cross-over of the 5-lipoxygenase and cyclooxygenase-2 pathways. They are formed by activated human leukocytes ex vivo, and, therefore, may be involved in regulation of the inflammatory response as autocrine or paracrine mediators. HKE and HKD are not commercially available and, so far, no method for their total chemical synthesis has been reported.

The Precise Structures and Stereochemistry of Trihydroxy-linoleates Esterified in Human and Porcine Epidermis and Their Significance in Skin Barrier Function: IMPLICATION OF AN EPOXIDE HYDROLASE IN THE TRANSFORMATIONS OF LINOLEATE.

Creation of an intact skin water barrier, a prerequisite for life on dry land, requires the lipoxygenase-catalyzed oxidation of the essential fatty acid linoleate, which is esterified to the ω-hydroxyl of an epidermis-specific ceramide. Oxidation of the linoleate moiety by lipoxygenases is proposed to facilitate enzymatic cleavage of the ester bond, releasing free ω-hydroxyceramide for covalent binding to protein, thus forming the corneocyte lipid envelope, a key component of the epidermal barrier. Herein, we report the transformations of esterified linoleate proceed beyond the initial steps of oxidation and epoxyalcohol synthesis catalyzed by the consecutive actions of 12R-LOX and epidermal LOX3.

Robust inhibitory effects of conjugated linolenic acids on a cyclooxygenase-related linoleate 10S-dioxygenase: Comparison with COX-1 and COX-2.

There are many reports of the anti-inflammatory, anti-cancer, and anti-atherosclerotic activities of conjugated linolenic acids (cLNA). They constitute a small percentage of fatty acids in the typical human diet, although up to 80% of the fatty acids in certain fruits such as pomegranate. In the course of studying a bacterial fatty acid dioxygenase (Nostoc linoleate 10S-DOX, an ancient relative of mammalian cyclooxygenases), we detected strong inhibitory activity in a commercial sample of linoleic acid.

A Catalase-related Hemoprotein in Coral Is Specialized for Synthesis of Short-chain Aldehydes: DISCOVERY OF P450-TYPE HYDROPEROXIDE LYASE ACTIVITY IN A CATALASE.

In corals a catalase-lipoxygenase fusion protein transforms arachidonic acid to the allene oxide 8R,9-epoxy-5,9,11,14-eicosatetraenoic acid from which arise cyclopentenones such as the prostanoid-related clavulones. Recently we cloned two catalase-lipoxygenase fusion protein genes (a and b) from the coral Capnella imbricata, form a being an allene oxide synthase and form b giving uncharacterized polar products (Lõhelaid, H., Teder, T.

Lipoxygenase-catalyzed transformation of epoxy fatty acids to hydroxy-endoperoxides: a potential P450 and lipoxygenase interaction.

Herein, we characterize a generally applicable transformation of fatty acid epoxides by lipoxygenase (LOX) enzymes that results in the formation of a five-membered endoperoxide ring in the end product. We demonstrated this transformation using soybean LOX-1 in the metabolism of 15,16-epoxy-α-linolenic acid, and murine platelet-type 12-LOX and human 15-LOX-1 in the metabolism of 14,15-epoxyeicosatrienoic acid (14,15-EET). A detailed examination of the transformation of the two enantiomers of 15,16-epoxy-α-linolenic acid by soybean LOX-1 revealed that the expected primary product, a 13S-hydroperoxy-15,16-epoxide, underwent a nonenzymatic transformation in buffer into a new derivative that was purified by HPLC and identified by UV, LC-MS, and ¹H-NMR as a 13,15-endoperoxy-16-hydroxy-octadeca-9,11-dienoic acid.