Insights from the x-ray crystal structure of coral 8R-lipoxygenase: Calcium activation via a C2-like domain and a structural basis of product chirality

Lipoxygenases (LOXs) catalyze the regio- and stereospecific dioxygenation of polyunsaturated membrane-embedded fatty acids. We report here the 3.2 Å resolution structure of 8R-LOX from the Caribbean sea whip coral Plexaura homomalla, a LOX isozyme with calcium dependence and the uncommon R chiral stereospecificity. Structural and spectroscopic analyses demonstrated calcium binding in a C2-like membrane-binding domain, illuminating the function of similar amino acids in calcium-activated mammalian 5-LOX, the key enzyme in the pathway to the pro-inflammatory leukotrienes. Mutation of Ca2+- ligating amino acids in 8R-LOX resulted not only in a diminished capacity to bind membranes, as monitored by fluorescence resonance energy transfer, but also in an associated loss of Ca2+-regulated enzyme activity. Moreover, a structural basis for R chiral specificity is also revealed; creation of a small oxygen pocket next to Gly428 (Ala in all S-LOX isozymes) promoted C-8 oxygenation with R chirality on the activated fatty acid substrate. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc

The importance of the lipoxygenase-hepoxilin pathway in the mammalian epidermal barrier

This review covers the background to discovery of the two key lipoxygenases (LOX) involved in epidermal barrier function, 12R-LOX and eLOX3, and our current views on their functioning. In the outer epidermis, their consecutive actions oxidize linoleic acid esterified in ω-hydroxy-ceramide to a hepoxilin-related derivative. The relevant background to hepoxilin and trioxilin biochemistry is briefly reviewed. We outline the evidence that linoleate in the ceramide is the natural substrate of the two LOX enzymes and our proposal for its importance in construction of the epidermal water barrier. Our hypothesis is that the oxidation promotes hydrolysis of the oxidized linoleate moiety from the ceramide. The resulting free ω-hydroxyl of the ω-hydroxyceramide is covalently bound to proteins on the surface of the corneocytes to form the corneocyte lipid envelope, a key barrier component. Understanding the role of the LOX enzymes and their hepoxilin products should provide rational approaches to ameliorative therapy for a number of the congenital ichthyoses involving compromised barrier function. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias

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. The major end product in human and porcine epidermis is a trihydroxy derivative, formed with a specificity that implicates participation of an epoxide hydrolase in converting epoxyalcohol to triol. Of the 16 possible triols arising from hydrolysis of 9,10-epoxy-13-hydroxy-octadecenoates, using LC-MS and chiral analyses, we identify and quantify specifically 9R,10S,13R-trihydroxy-11E-octadecenoate as the single major triol esterified in porcine epidermis and the same isomer with lesser amounts of its 10R diastereomer in human epidermis. The 9R,10S,13R-triol is formed by SN2 hydrolysis of the 9R,10R-epoxy-13R-hydroxy-octadecenoate product of the LOX enzymes, a reaction specificity characteristic of epoxide hydrolase. The high polarity of triol over the primary linoleate products enhances the concept that the oxidations disrupt corneocyte membrane lipids, promoting release of free ω-hydroxyceramide for covalent binding to protein and sealing of the waterproof barrier

Upregulation of 8-Lipoxygenase in the Dermatitis of IκB-α-Deficient Mice

Neonatal mice deficient in IκB-α, an inhibitor of the ubiquitous transcription factor NF-κB, develop severe and widespread dermatitis shortly after birth. In humans, inflammatory skin disorders such as psoriasis are associated with accumulation in the skin of the unusual arachidonic acid metabolite 12R-hydroxyeicosatetraenoic acid (12R-HETE), a product of the enzyme 12R-lipoxygenase. To examine the etiology of the murine IκB-α-deficient skin phenotype, we investigated the expression of lipoxygenases and the metabolism of exogenous arachidonic acid in the skin. In the IκB-α-deficient animals, the major lipoxygenase metabolite was 8S-HETE, formed together with a minor amount of 12S-HETE; 12R-HETE synthesis was undetectable. Skin from the wild-type littermates formed 12S-HETE as the almost exclusive lipoxygenase metabolite. Upregulation of 8S-lipoxygenase (8-LOX) in IκB-α-deficient mice was confirmed at the transcriptional and translational level using ribonuclease protection assay and western analysis. In immunohistochemical studies, increased expression of 8-LOX was detected in the stratum granulosum of the epidermis. In the stratum granulosum, 8-LOX may be involved in the terminal differentiation of keratinocytes. Although mouse 8S-lipoxygenase and human 12R-lipoxygenase are not ortholog genes, we speculate that in mouse and humans the two different enzymes may fulfill equivalent functions in the progression of inflammatory dermatoses

Crystal structure of a lipoxygenase in complex with substrate: The arachidonic acid-binding site of 8R-lipoxygenase

Lipoxygenases (LOX) play critical roles in mammalian biology in the generation of potent lipid mediators of the inflammatory response; consequently, they are targets for the development of isoform-specific inhibitors. The regio- and stereo-specificity of the oxygenation of polyunsaturated fatty acids by the enzymes is understood in terms of the chemistry, but structural observation of the enzyme-substrate interactions is lacking. Although several LOX crystal structures are available, heretofore the rapid oxygenation of bound substrate has precluded capture of the enzyme-substrate complex, leaving a gap between chemical and structural insights. In this report, we describe the 2.0 Å resolution structure of 8R-LOX in complex with arachidonic acid obtained under anaerobic conditions. Subtle rearrangements, primarily in the side chains of three amino acids, allow binding of arachidonic acid in a catalytically competent conformation. Accompanying experimental work supports a model in which both substrate tethering and cavity depth contribute to positioning the appropriate carbon at the catalytic machinery

The structure of human 5-lipoxygenase

The synthesis of both proinflammatory leukotrienes and anti-inflammatory lipoxins requires the enzyme 5-lipoxygenase (5-LOX). 5-LOX activity is short-lived, apparently in part because of an intrinsic instability of the enzyme. We identified a 5-LOX-specific destabilizing sequence that is involved in orienting the carboxyl terminus, which binds the catalytic iron. Here, we report the crystal structure at 2.4 angstrom resolution of human 5-LOX stabilized by replacement of this sequence