Structure of a model lipid membrane oxidized by human 15-lipoxygenase-2.

Enzyme-mediated lipid oxidation is an important regulatory event in cell signaling, with oxidized lipids being potent signaling molecules that can illicit dramatic changes in cell behavior. For example, peroxidation of an arachidonoyl poly-unsaturated fatty acid by the human enzyme 15-lipoxygenase-2 (15-LOX-2) has been associated with formation of atherosclerotic plaques. Previous work on synthetically oxidized membranes has shown that oxidized lipid tails will change their conformation to facilitate interactions between the peroxide group and the lipid headgroups.

Cannabidiol acts as molecular switch in innate immune cells to promote the biosynthesis of inflammation-resolving lipid mediators.

Cannabinoids are phytochemicals from cannabis with anti-inflammatory actions in immune cells. Lipid mediators (LM), produced from polyunsaturated fatty acids (PUFA), are potent regulators of the immune response and impact all stages of inflammation. How cannabinoids influence LM biosynthetic networks is unknown.

Allosteric Activation of 15-Lipoxygenase-1 by Boswellic Acid Induces the Lipid Mediator Class Switch to Promote Resolution of Inflammation.

Specialized pro-resolving mediators (SPM), primarily produced in innate immune cells, exert crucial bioactions for resolving inflammation. Among various lipoxygenases (LOX), 15-LOX-1 is key for SPM biosynthesis, but cellular activation principles of 15-LOX-1 are unexplored. It was shown that 3-O-acetyl-11-keto-β-boswellic acid (AKBA) shifts 5-LOX regiospecificity from 5- to 12-lipoxygenation products.

Structures of Leukotriene Biosynthetic Enzymes and Development of New Therapeutics.

Leukotrienes are potent immune-regulating lipid mediators with patho-genic roles in inflammatory and allergic diseases, particularly asthma. These autacoids also contribute to low-grade inflammation, a hallmark of cardiovascular, neurodegenerative, metabolic, and tumor diseases. Biosynthesis of leukotrienes involves release and oxidative metabolism of arachidonic acid and proceeds via a set of cytosolic and integral membrane enzymes that are typically expressed by cells of the innate immune system.

Helical remodeling augments 5-lipoxygenase activity in the synthesis of proinflammatory mediators.

The synthesis of proinflammatory leukotrienes implicated in asthma, allergic rhinitis, and atherosclerosis is initiated by the enzyme 5-lipoxygenase (5-LOX). The crystal structure of human Stable-5-LOX revealed a conformation where the catalytic iron was inaccessible to bulk solvent as two aromatic residues on a conserved helix-α2 (Hα2) plugged the substrate access portal. Whether 5-LOX can also adopt a more open conformation has not been resolved.

Untangling the web of 5-lipoxygenase-derived products from a molecular and structural perspective: The battle between pro- and anti-inflammatory lipid mediators.

Arachidonic acid (AA) is the precursor to leukotrienes (LT), potent mediators of the inflammatory response. In the 35 + years since cysteinyl-LTs were reported to mediate antigen-induced constriction of bronchi in tissue from asthma patients, numerous cellular responses evoked by the LTs, such as chemoattraction and G protein-coupled receptor (GPCR) activation, have been elucidated and revealed a potential for 5-lipoxygenase (5-LOX) as a promising drug target that goes beyond asthma. We describe herein early work identifying 5-LOX as the key enzyme that initiates LT biosynthesis and the discovery of its membrane-embedded helper protein required to execute the two-step reaction that transforms AA to the progenitor leukotriene A (LTA).

Anti-inflammatory celastrol promotes a switch from leukotriene biosynthesis to formation of specialized pro-resolving lipid mediators.

The pentacyclic triterpenoid quinone methide celastrol (CS) from Tripterygium wilfordii Hook. F. effectively ameliorates inflammation with potential as therapeutics for inflammatory diseases.

Structural and mechanistic insights into 5-lipoxygenase inhibition by natural products.

Leukotrienes (LT) are lipid mediators of the inflammatory response that are linked to asthma and atherosclerosis. LT biosynthesis is initiated by 5-lipoxygenase (5-LOX) with the assistance of the substrate-binding 5-LOX-activating protein at the nuclear membrane. Here, we contrast the structural and functional consequences of the binding of two natural product inhibitors of 5-LOX.

Chloride to the rescue.

On the fiftieth anniversary of the discovery of the Ser-His-Asp catalytic triad, perhaps the most unusual variation on the textbook classic is described: An incomplete catalytic triad in a hydrolase is rescued by a chloride ion (Fig. 1). Structural and functional data provide compelling evidence that the active site of a phospholipase from employs the anion in place of the commonly observed Asp, reminding us that even well-trodden scientific ground has surprises in store.

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.

A 5‑lipoxygenase-specific sequence motif impedes enzyme activity and confers dependence on a partner protein.

Leukotrienes (LT) are lipid mediators of the inflammatory response that play key roles in diseases such as asthma and atherosclerosis. The precursor leukotriene A (LTA) is synthesized from arachidonic acid (AA) by 5‑lipoxygenase (5-LOX), a membrane-associated enzyme, with the help of 5‑lipoxygenase-activating protein (FLAP), a nuclear transmembrane protein. In lipoxygenases the main chain carboxylate of the C-terminus is a ligand for the non-heme iron and thus part of the catalytic center.

Expression of an 8R-Lipoxygenase From the Coral Plexaura homomalla.

Methods are presented for the use of the coral 8R-lipoxygenase from the Caribbean sea whip coral Plexaura homomalla as a model enzyme for structural studies of animal lipoxygenases. The 8R-lipoxygenase is remarkably stable and can be stored at 4°C for 3 months with virtually no loss of activity. In addition, an engineered "pseudo wild-type" enzyme is soluble in the absence of detergents, which helps facilitate the preparation of enzyme:substrate complexes.

Identification and optimization of soluble epoxide hydrolase inhibitors with dual potency towards fatty acid amide hydrolase.

Multi-target inhibitors have become increasing popular as a means to leverage the advantages of poly-pharmacology while simplifying drug delivery. Here, we describe dual inhibitors for soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH), two targets known to synergize when treating inflammatory and neuropathic pain. The structure activity relationship (SAR) study described herein initially started with t-TUCB (trans-4-[4-(3-trifluoromethoxyphenyl-l-ureido)-cyclohexyloxy]-benzoic acid), a potent sEH inhibitor that was previously shown to weakly inhibit FAAH.

Structural basis of antifreeze activity of a bacterial multi-domain antifreeze protein.

Antifreeze proteins (AFPs) enhance the survival of organisms inhabiting cold environments by affecting the formation and/or structure of ice. We report the crystal structure of the first multi-domain AFP that has been characterized. The two ice binding domains are structurally similar.

The Thr-His Connection on the Distal Heme of Catalase-Related Hemoproteins: A Hallmark of Reaction with Fatty Acid Hydroperoxides.

This review focuses on a group of heme peroxidases that retain the catalase fold in structure, yet show little or no reaction with hydrogen peroxide. Instead of having a role in oxidative defense, these enzymes are involved in secondary metabolite biosynthesis. The prototypical enzyme is catalase-related allene oxide synthase, an enzyme that converts a specific fatty acid hydroperoxide to the corresponding allene oxide (epoxide).

Membrane-dependent Activities of Human 15-LOX-2 and Its Murine Counterpart: IMPLICATIONS FOR MURINE MODELS OF ATHEROSCLEROSIS.

The enzyme encoded by the ALOX15B gene has been linked to the development of atherosclerotic plaques in humans and in a mouse model of hypercholesterolemia. In vitro, these enzymes, which share 78% sequence identity, generate distinct products from their substrate arachidonic acid: the human enzyme, a 15-S-hydroperoxy product; and the murine enzyme, an 8-S-product. We probed the activities of these enzymes with nanodiscs as membrane mimics to determine whether they can access substrate esterified in a bilayer and characterized their activities at the membrane interface.

5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation.

Leukotrienes (LTs) are proinflammatory lipid mediators formed from arachidonic acid in a 2-step reaction catalyzed by 5-lipoxygenase (5-LOX) requiring the formation of 5-HPETE [5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid] and its subsequent transformation to LTA4 5-LOX is thought to receive arachidonic acid from the nuclear membrane-embedded 5-LOX-activating protein (FLAP). The crystal structure of 5-LOX revealed an active site concealed by F177 and Y181 (FY cork). We examined the influence of the FY cork on 5-LOX activity and membrane binding in HEK293 cells in the absence and presence of FLAP.

Identification of the Substrate Access Portal of 5-Lipoxygenase.

The overproduction of inflammatory lipid mediators derived from arachidonic acid contributes to asthma and cardiovascular diseases, among other pathologies. Consequently, the enzyme that initiates the synthesis of pro-inflammatory leukotrienes, 5-lipoxygenase (5-LOX), is a target for drug design. The crystal structure of 5-LOX revealed a fully encapsulated active site; thus the point of substrate entry is not known.

The structural basis for specificity in lipoxygenase catalysis.

Many intriguing facets of lipoxygenase (LOX) catalysis are open to a detailed structural analysis. Polyunsaturated fatty acids with two to six double bonds are oxygenated precisely on a particular carbon, typically forming a single chiral fatty acid hydroperoxide product. Molecular oxygen is not bound or liganded during catalysis, yet it is directed precisely to one position and one stereo configuration on the reacting fatty acid.

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.

Optimized inhibitors of soluble epoxide hydrolase improve in vitro target residence time and in vivo efficacy.

Diabetes is affecting the life of millions of people. A large proportion of diabetic patients suffer from severe complications such as neuropathic pain, and current treatments for these complications have deleterious side effects. Thus, alternate therapeutic strategies are needed.

Folate in demethylation: the crystal structure of the rat dimethylglycine dehydrogenase complexed with tetrahydrofolate.

Dimethylglycine dehydrogenase (DMGDH) is a mammalian mitochondrial enzyme which plays an important role in the utilization of methyl groups derived from choline. DMGDH is a flavin containing enzyme which catalyzes the oxidative demethylation of dimethylglycine in vitro with the formation of sarcosine (N-methylglycine), hydrogen peroxide and formaldehyde. DMGDH binds tetrahydrofolate (THF) in vivo, which serves as an acceptor of formaldehyde and in the cell the product of the reaction is 5,10-methylenetetrahydrofolate instead of formaldehyde.

Crystal structure of the histone lysine specific demethylase LSD1 complexed with tetrahydrofolate.

An important epigenetic modification is the methylation/demethylation of histone lysine residues. The first histone demethylase to be discovered was a lysine-specific demethylase 1, LSD1, a flavin containing enzyme which carries out the demethylation of di- and monomethyllysine 4 in histone H3. The removed methyl groups are oxidized to formaldehyde.

The structure of human 15-lipoxygenase-2 with a substrate mimic.

Atherosclerosis is associated with chronic inflammation occurring over decades. The enzyme 15-lipoxygenase-2 (15-LOX-2) is highly expressed in large atherosclerotic plaques, and its activity has been linked to the progression of macrophages to the lipid-laden foam cells present in atherosclerotic plaques. We report here the crystal structure of human 15-LOX-2 in complex with an inhibitor that appears to bind as a substrate mimic.