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.

The structure of the SufS-SufE complex reveals interactions driving protected persulfide transfer in iron-sulfur cluster biogenesis.

Fe-S clusters are critical cofactors for redox chemistry in all organisms. The cysteine desulfurase, SufS, provides sulfur in the SUF Fe-S cluster bioassembly pathway. SufS is a dimeric, pyridoxal 5'-phosphate-dependent enzyme that uses cysteine as a substrate to generate alanine and a covalent persulfide on an active site cysteine residue.

The structure of the SufS-SufE complex reveals interactions driving protected persulfide transfer in iron-sulfur cluster biogenesis.

Fe-S clusters are critical cofactors for redox chemistry in all organisms. The cysteine desulfurase, SufS, provides sulfur in the SUF Fe-S cluster bioassembly pathway. SufS is a dimeric, PLP-dependent enzyme that uses cysteine as a substrate to generate alanine and a covalent persulfide on an active site cysteine residue.

Identification of the Thermal Activation Network in Human 15-Lipoxygenase-2: Divergence from Plant Orthologs and Its Relationship to Hydrogen Tunneling Activation Barriers.

The oxidation of polyunsaturated fatty acids by lipoxygenases (LOXs) is initiated by a C-H cleavage step in which the hydrogen atom is transferred quantum mechanically (i.e., via tunneling).

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.

Investigating membrane-binding properties of lipoxygenases using surface plasmon resonance.

Lipoxygenases (LOXs) catalyze the oxidation of polyunsaturated fatty acids and synthesize oxylipin products that drive important cellular signaling processes in plants and animals. While there has been indirect evidence presented for the interaction of mammalian LOXs with membranes, a quantitative study of the molecular details of LOX-membrane interactions is lacking. Here, we mimicked biological membranes using surface plasmon resonance (SPR) sensor chips derivatized with 2-D planar lipophilic anchors (2D LP) to capture liposomes of varying phospholipid compositions that self-assemble into lipid bilayers on the SPR chip.

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.

Inhibition of Fosfomycin Resistance Protein FosB from Gram-Positive Pathogens by Phosphonoformate.

The Gram-positive pathogen is a leading cause of antimicrobial resistance related deaths worldwide. Like many pathogens with multidrug-resistant strains, contains enzymes that confer resistance through antibiotic modification(s). One such enzyme present in is FosB, a Mn-dependent l-cysteine or bacillithiol (BSH) transferase that inactivates the antibiotic fosfomycin.

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.

Characterization of the genomically encoded fosfomycin resistance enzyme from .

belongs to a group of rapidly growing mycobacteria (RGM) and accounts for approximately 65-80% of lung disease caused by RGM. It is highly pathogenic and is considered the prominent Mycobacterium involved in pulmonary infection in patients with cystic fibrosis and chronic pulmonary disease (CPD). FosM is a putative 134 amino acid fosfomycin resistance enzyme from subsp.

X-ray structure of chromium(III)-containing transferrin: First structure of a physiological Cr(III)-binding protein.

Transferrin, the Fe(III) transport protein in mammalian blood, has been suggested to also serve as a Cr(III) transporter and as part of a Cr(III) detoxification system; however, the structure of the metal-binding sites has never been fully elucidated with bound Cr(III). Chromium(III)-transferrin was crystallized in the presence of the synergistic anion malonate. In the crystals, the protein exists with a closed C-terminal lobe containing a Cr(III) ion and an open, unoccupied N-terminal lobe.

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.

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.

Structural basis of arrestin-3 activation and signaling.

A unique aspect of arrestin-3 is its ability to support both receptor-dependent and receptor-independent signaling. Here, we show that inositol hexakisphosphate (IP) is a non-receptor activator of arrestin-3 and report the structure of IP-activated arrestin-3 at 2.4-Å resolution.

Structure of a calcium-dependent 11R-lipoxygenase suggests a mechanism for Ca2+ regulation.

Lipoxygenases (LOXs) are a key part of several signaling pathways that lead to inflammation and cancer. Yet, the mechanisms of substrate binding and allosteric regulation by the various LOX isoforms remain speculative. Here we report the 2.

Conversion of human 5-lipoxygenase to a 15-lipoxygenase by a point mutation to mimic phosphorylation at Serine-663.

The enzyme 5-lipoxygenase (5-LOX) initiates biosynthesis of the proinflammatory leukotriene lipid mediators and, together with 15-LOX, is also required for synthesis of the anti-inflammatory lipoxins. The catalytic activity of 5-LOX is regulated through multiple mechanisms, including Ca(2+)-targeted membrane binding and phosphorylation at specific serine residues. To investigate the consequences of phosphorylation at S663, we mutated the residue to the phosphorylation mimic Asp, providing a homogenous preparation suitable for catalytic and structural studies.

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.

Location, location, location: compartmentalization of early events in leukotriene biosynthesis.

Leukotrienes (LTs), derived from arachidonic acid (AA) released from the membrane by the action of phospholipase A(2), are potent lipid mediators of the inflammatory response. In 1983, Dahlén et al. demonstrated that LTC(4), LTD(4), and LTE(4) mediate antigen-induced constriction of bronchi in tissue obtained from subjects with asthma (Dahlén, S.

The 1.85 A structure of an 8R-lipoxygenase suggests a general model for lipoxygenase product specificity.

Lipoxygenases (LOX) play pivotal roles in the biosynthesis of leukotrienes and other biologically active eicosanoids derived from arachidonic acid. A mechanistic understanding of substrate recognition, when lipoxygenases that recognize the same substrate generate different products, can be used to help guide the design of enzyme-specific inhibitors. We report here the 1.

A covalent linker allows for membrane targeting of an oxylipin biosynthetic complex.

A naturally occurring bifunctional protein from Plexaura homomalla links sequential catalytic activities in an oxylipin biosynthetic pathway. The C-terminal lipoxygenase (LOX) portion of the molecule catalyzes the transformation of arachidonic acid (AA) to the corresponding 8 R-hydroperoxide, and the N-terminal allene oxide synthase (AOS) domain promotes the conversion of the hydroperoxide intermediate to the product allene oxide (AO). Small-angle X-ray scattering data indicate that in the absence of a covalent linkage the two catalytic domains that transform AA to AO associate to form a complex that recapitulates the structure of the bifunctional protein.

Improving protein crystal quality by selective removal of a Ca(2+)-dependent membrane-insertion loop.

Lipoxygenases (LOXs) catalyze the regiospecific and stereospecific dioxygenation of polyunsaturated membrane-embedded fatty acids. A Ca(2+)-dependent membrane-binding function was localized to the amino-terminal C2-like domain of 8R-lipoxygenase (8R-LOX) from the soft coral Plexaura homomalla. The 3.