The leukotrienes are important mediators in asthma as well as in other inflammatory and allergic disorders. 5-Lipoxygenase (5-LO 1 ; arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) catalyzes two initial steps in the cellular production of leukotrienes. Thus, 5-LO converts arachidonic acid into 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid and subsequently into the unstable epoxide leukotriene A 4 , which, in turn, is the precursor of the biologically active leukotrienes B 4 , C 4 , D 4 , and E 4 (1). Leukotriene B 4 stimulates adherence of leukocytes to the vessel wall and is a potent chemotactic agent for these cells. The cysteinyl leukotrienes C 4 , D 4 , and E 4 increase vascular permeability and are effective constrictors of bronchial smooth muscle. 5-LO also participates in the formation of lipoxins, another group of arachidonate-derived bioactive lipids that are implicated in inflammatory and vascular events (2).Calcium is a well known 5-LO activator (for reviews, see Refs. 3-5). In brief, stimuli that elevate the intracellular Ca 2ϩ levels were shown to induce cellular 5-LO activity, and several reports have described Ca 2ϩ -induced translocation of 5-LO from the cytosol to cellular membranes. More detailed analyses showed an association primarily with the nuclear envelope, where the membrane-bound 5-LO-activating protein (FLAP) is also found and where the substrate arachidonic acid can be released from membrane lipids by cytosolic phospholipase A 2 (cPLA 2 ). The stimulatory effect of Ca 2ϩ is evident also for purified 5-LO. The basal enzyme activity, which is observed in the presence of a membrane fraction or lipids, increases up to 10-fold if micromolar concentrations of Ca 2ϩ are included in the assay mixture. 5-LO catalysis has been shown to occur at the lipid/water interface, and Ca 2ϩ -dependent binding of 5-LO to phospholipid vesicles has been reported. By several experimental approaches, we have recently demonstrated that 5-LO binds Ca 2ϩ in a reversible manner (3). A K d close to 6 M was determined by equilibrium dialysis, and the stoichiometry of maximum binding averaged around two Ca 2ϩ ions/5-LO molecule. We also showed that binding of calcium increased the hydrophobicity of 5-LO. Thus, a present conception is that calcium stimulates 5-LO activity and leukotriene production by promoting membrane association.The first structural determination of a mammalian 15-lipoxygenase (6) revealed that, similar to soybean lipoxygenases (7-9), it is composed of two major domains: a C-terminal domain containing the catalytic site and an N-terminal ␤-barrel domain. It seems reasonable that this is the overall structure also for 5-LO. The capability of 5-LO to bind more than one calcium ion and the calcium-dependent binding to phospholipids make 5-LO functionally similar to a group of calciumbinding proteins known as C2 domain proteins. The C2 domain is a conserved structural motif that forms an eight-stranded anti-parallel ␤-sandwich, and C2 domains have been identified in some 70 membrane-...

Cysteinyl leukotrienes are key mediators in inflammation and have an important role in acute and chronic inflammatory diseases of the cardiovascular and respiratory systems, in particular bronchial asthma. In the biosynthesis of cysteinyl leukotrienes, conversion of arachidonic acid forms the unstable epoxide leukotriene A4 (LTA4). This intermediate is conjugated with glutathione (GSH) to produce leukotriene C4 (LTC4) in a reaction catalysed by LTC4 synthase: this reaction is the key step in cysteinyl leukotriene formation. Here we present the crystal structure of the human LTC4 synthase in its apo and GSH-complexed forms to 2.00 and 2.15 A resolution, respectively. The structure reveals a homotrimer, where each monomer is composed of four transmembrane segments. The structure of the enzyme in complex with substrate reveals that the active site enforces a horseshoe-shaped conformation on GSH, and effectively positions the thiol group for activation by a nearby arginine at the membrane-enzyme interface. In addition, the structure provides a model for how the omega-end of the lipophilic co-substrate is pinned at one end of a hydrophobic cleft, providing a molecular 'ruler' to align the reactive epoxide at the thiol of glutathione. This provides new structural insights into the mechanism of LTC4 formation, and also suggests that the observed binding and activation of GSH might be common for a family of homologous proteins important for inflammatory and detoxification responses.

We have recently identified coactosin-like protein (CLP) in a yeast two-hybrid screen using 5-lipoxygenase (5LO) as a bait. In this report, we demonstrate a direct interaction between 5LO and CLP. 5LO associated with CLP, which was expressed as a glutathione S-transferase fusion protein, in a dose-dependent manner. Coimmunoprecipitation experiments using epitope-tagged 5LO and CLP proteins transiently expressed in human embryonic kidney 293 cells revealed the presence of CLP in 5LO immunoprecipitates. In reciprocal experiments, 5LO was detected in CLP immunoprecipitates. Non-denaturing polyacrylamide gel electrophoresis and cross-linking experiments showed that 5LO binds CLP in a 1:1 molar stoichiometry in a Ca 2؉ -independent manner. Site-directed mutagenesis suggested an important role for lysine 131 of CLP in mediating 5LO binding. In view of the ability of CLP to bind 5LO and filamentous actin (F-actin), we determined whether CLP could physically link 5LO to actin filaments. However, no F-actin-CLP⅐5LO ternary complex was observed. In contrast, 5LO appeared to compete with F-actin for the binding of CLP. Moreover, 5LO was found to interfere with actin polymerization. Our results indicate that the 5LO-CLP and CLP-F-actin interactions are mutually exclusive and suggest a modulatory role for 5LO in actin dynamics. 5-Lipoxygenase (5LO)1 is of central importance in cellular leukotriene (LT) synthesis. This enzyme converts arachidonic acid released from the membranes by the cytosolic phospholipase A 2 into 5(S)-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and subsequently into the epoxide intermediate LTA 4 (1). LTA 4 is further metabolized into LTB 4 by the LTA 4 hydrolase or into LTC 4 through the action of the LTC 4 synthase. LTC 4 is then sequentially degraded into LTD 4 and LTE 4 .Whereas LTB 4 exerts potent stimulatory effects on various leukocyte functions, including chemotaxis, adhesion, degranulation, and aggregation, the cysteinyl-LTs (LTC 4 , LTD 4 , and LTE 4 ) are known to contract airway smooth muscle, increase vascular permeability, and promote mucus secretion (2). 5LO and LTs are, therefore, key components involved in inflammatory disorders, including arthritis, asthma, and allergic reactions.Recently, novel modulatory mechanisms determining cellular 5LO activity were identified. 5LO is phosphorylated by p38 mitogen-activated protein kinase-activated protein (MAPKAP) kinases prepared from stimulated myeloid cells (3). In addition, Mg 2ϩ increases 5LO activity in vitro (4). Furthermore, a stimulatory Ca 2ϩ binding site has been localized in the N-terminal domain of 5LO, that may function as a C2 domain in the calcium regulation of 5LO catalytic activity (5). C2 domains have also been shown to mediate protein-protein interactions (6).Additional lines of evidence indicate that cellular 5LO activity and distribution is regulated by interaction with other proteins. For example, the subcellular distribution of 5LO differs among cell types and changes in response to various stimuli. In particular...

5-Lipoxygenase (5LO) catalyzes the first two steps in the biosynthesis of leukotrienes and lipoxins and has therefore become an important target for pharmacological treatment of inflammatory disorders. Binding of calcium to 5LO was shown using several different approaches. Human recombinant enzyme was expressed in E. coli and purified. Association of Ca2+ to 5LO was demonstrated by a calcium-induced mobility shift in gel electrophoresis, by calcium overlay, by gel filtration in the presence of calcium, and by equilibrium dialysis. The two latter methods also showed that calcium binds reversibly to 5LO. Equilibrium dialysis gave a Kd close to 6 microM; the stoichiometry of maximum calcium binding seemed to average around two Ca2+ per 5LO. Similar results were obtained when 5LO was inactivated during equilibrium dialysis, indicating that the calcium binding site(s) is (are) different from the active site. By Triton X-114 partitioning, it was confirmed that calcium increases the hydrophobicity of 5LO.

Mg2+ gave dose-dependent activation of 5-lipoxygenase (5LO) in vitro. As for Ca2+, the activation depended on the presence of phosphatidylcholine (PC) vesicles, and the activation response was different at various combinations of arachidonate and PC. Stimulation of 5LO activity was observed with Mg2+ concentrations of 0.1-1 mM, similar to the concentration range of free Mg2+ in mammalian cells. However, to observe a clear increase in 5LO hydrophobicity, a higher concentration of Mg2+ (4 mM) was required, and at this concentration also 5LO activation was optimal. Combinations of Mg2+ with ATP (containing free Mg2+ and MgATP2- complex) gave better activation of 5LO than either agent alone. This effect of Mg2+ (and ATP) could be of interest in relation to basal 5LO activity in cells not subjected to a particular stimulus.