RESUMO
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. However, this phenomenon has not been directly observed for a lipid membrane that has undergone enzyme-catalyzed oxidation. In this study, we report on the structure of a model lipid membrane before and after oxidation by 15-LOX-2. A model lipid membrane monolayer at the air-liquid interface was constructed from 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (SAPC) in a Langmuir trough, and X-ray reflectivity measurements were conducted to determine the electron density profile of the system. Exposure to 15-LOX-2 caused a dramatic change in the SAPC structure, namely a blurred distinction between the lipid tail/head layers and shortening of the average lipid tail length by â¼3 Å. The electron density profile of the oxidized SAPC monolayer is similar to that of a synthetically oxidized substrate mimic. Overall, this reported observation of an enzymatically-oxidized membrane structure in situ is helping to bridge a gap in the literature between structural studies on synthetically oxidized membranes and cellular studies aiming to understand physiological responses.
RESUMO
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. SufS enzymes are activated by an accessory transpersulfurase protein, either SufE or SufU depending on the organism, which accepts the persulfide product and delivers it to downstream partners for Fe-S assembly. Here, using E. coli proteins, we present the first X-ray crystal structure of a SufS/SufE complex. There is a 1:1 stoichiometry with each monomeric unit of the EcSufS dimer bound to one EcSufE subunit, though one EcSufE is rotated â¼7° closer to the EcSufS active site. EcSufE makes clear interactions with the α16 helix of EcSufS and site-directed mutants of several α16 residues were deficient in EcSufE binding. Analysis of the EcSufE structure showed a loss of electron density at the EcSufS/EcSufE interface for a flexible loop containing the highly conserved residue R119. An R119A EcSufE variant binds EcSufS but is not active in cysteine desulfurase assays and fails to support Fe-S cluster bioassembly in vivo. 35S-transfer assays suggest that R119A EcSufE can receive a persulfide, suggesting the residue may function in a release mechanism. The structure of the EcSufS/EcSufE complex allows for comparison with other cysteine desulfurases to understand mechanisms of protected persulfide transfer across protein interfaces.
RESUMO
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. SufS enzymes are activated by an accessory transpersulfurase protein, either SufE or SufU depending on the organism, which accepts the persulfide product and delivers it to downstream partners for Fe-S assembly. Here, using E. coli proteins, we present the first X-ray crystal structure of a SufS/SufE complex. There is a 1:1 stoichiometry with each monomeric unit of the EcSufS dimer bound to one EcSufE subunit, though one EcSufE is rotated ~7° closer to the EcSufS active site. EcSufE makes clear interactions with the α16 helix of EcSufS and site-directed mutants of several α16 residues were deficient in EcSufE binding. Analysis of the EcSufE structure showed a loss of electron density at the EcSufS/EcSufE interface for a flexible loop containing the highly conserved residue R119. An R119A EcSufE variant binds EcSufS but is not active in cysteine desulfurase assays and fails to support Fe-S cluster bioassembly in vivo. 35S-transfer assays suggest that R119A EcSufE can receive a persulfide, suggesting the residue may function in a release mechanism. The structure of the EcSufS/EcSufE complex allows for comparison with other cysteine desulfurases to understand mechanisms of protected persulfide transfer across protein interfaces.
RESUMO
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). In these reactions, protein thermal motions facilitate the conversion of ground-state enzyme-substrate complexes to tunneling-ready configurations and are thus important for transferring energy from the solvent to the active site for the activation of catalysis. In this report, we employed temperature-dependent hydrogen-deuterium exchange mass spectrometry (TDHDX-MS) to identify catalytically linked, thermally activated peptides in a representative animal LOX, human epithelial 15-LOX-2. TDHDX-MS of wild-type 15-LOX-2 was compared to two active site mutations that retain structural stability but have increased activation energies (Ea) of catalysis. The Ea value of one variant, V427L, is implicated to arise from suboptimal substrate positioning by increased active-site side chain rotamer dynamics, as determined by X-ray crystallography and ensemble refinement. The resolved thermal network from the comparative Eas of TDHDX-MS between wild-type and V426A is localized along the front face of the 15-LOX-2 catalytic domain. The network contains a clustering of isoleucine, leucine, and valine side chains within the helical peptides. This thermal network of 15-LOX-2 is different in location, area, and backbone structure compared to a model plant lipoxygenase from soybean that exhibits a low Ea value of catalysis compared to the human ortholog. The presented data provide insights into the divergence of thermally activated protein motions in plant and animal LOXs and their relationships to the enthalpic barriers for facilitating hydrogen tunneling.
RESUMO
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. Here, we reveal cannabidiol (CBD) as a potent LM class-switching agent that stimulates the production of specialized pro-resolving mediators (SPMs) but suppresses pro-inflammatory eicosanoid biosynthesis. Detailed metabololipidomics analysis in human monocyte-derived macrophages showed that CBD (i) upregulates exotoxin-stimulated generation of SPMs, (ii) suppresses 5-lipoxygenase (LOX)-mediated leukotriene production, and (iii) strongly induces SPM and 12/15-LOX product formation in resting cells by stimulation of phospholipase A2-dependent PUFA release and through Ca2+-independent, allosteric 15-LOX-1 activation. Finally, in zymosan-induced murine peritonitis, CBD increased SPM and 12/15-LOX products and suppressed pro-inflammatory eicosanoid levels in vivo. Switching eicosanoid to SPM production is a plausible mode of action of CBD and a promising inflammation-resolving strategy.
Assuntos
Canabidiol , Humanos , Animais , Camundongos , Canabidiol/farmacologia , Inflamação/tratamento farmacológico , Eicosanoides , Macrófagos , Ácidos Graxos Insaturados/farmacologia , Imunidade InataRESUMO
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. The sensor chip surfaces were then used to investigate the membrane-binding properties of model LOX enzymes. SPR binding assays displayed reproducible and stable liposome capture to the sensor chip surface that allowed for the detailed characterization of LOX-membrane interactions. Our studies demonstrate a calcium-dependence for the membrane binding activities of coral 8R-LOX and human 15-LOX-2. Furthermore, our data confirm the importance of key membrane insertion loop residues in each of these LOX enzymes for membrane binding activity. Experiments utilizing model plant and human LOXs reveal differences in membrane-binding specificities. Our study establishes and validates a robust SPR-based platform using 2D LP sensor chips that allows for the detailed study of LOX-membrane interactions under different experimental conditions, including altered membrane compositions. Collectively, this investigation improves our overall understanding of LOX-membrane interaction properties, and our SPR-based approach holds potential for future use in the development of LOX-based therapeutics.
Assuntos
Lipoxigenases , Ressonância de Plasmônio de Superfície , Animais , Humanos , Bicamadas Lipídicas , Membrana Celular , Lipossomos , MamíferosRESUMO
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-ß-boswellic acid (AKBA) shifts 5-LOX regiospecificity from 5- to 12-lipoxygenation products. Here, it is demonstrated that AKBA additionally activates cellular 15-LOX-1 via an allosteric site accomplishing robust SPM formation in innate immune cells, particularly in M2 macrophages. Compared to ionophore, AKBA-induced LOX activation is Ca2+ - and phosphorylation-independent, with modest induction of 5-LOX products. AKBA docks into a groove between the catalytic and regulatory domains of 15-LOX-1 interacting with R98; replacement of R98 by alanine abolishes AKBA-induced 15-LOX product formation in HEK293 cells. In zymosan-induced murine peritonitis, AKBA strikingly elevates SPM levels and promotes inflammation resolution. Together, targeted allosteric modulation of LOX activities governs SPM formation and offers new concepts for inflammation resolution pharmacotherapy.
Assuntos
Araquidonato 15-Lipoxigenase , Lipoxigenase , Humanos , Camundongos , Animais , Regulação Alostérica , Células HEK293 , Inflamação/tratamento farmacológico , Lipídeos , Receptores Depuradores Classe ERESUMO
The Gram-positive pathogen Staphylococcus aureus is a leading cause of antimicrobial resistance related deaths worldwide. Like many pathogens with multidrug-resistant strains, S. aureus contains enzymes that confer resistance through antibiotic modification(s). One such enzyme present in S. aureus is FosB, a Mn2+-dependent l-cysteine or bacillithiol (BSH) transferase that inactivates the antibiotic fosfomycin. fosB gene knockout experiments show that the minimum inhibitory concentration (MIC) of fosfomycin is significantly reduced when the FosB enzyme is not present. This suggests that inhibition of FosB could be an effective method to restore fosfomycin activity. We used high-throughput in silico-based screening to identify small-molecule analogues of fosfomycin that inhibited thiol transferase activity. Phosphonoformate (PPF) was a top hit from our approach. Herein, we have characterized PPF as a competitive inhibitor of FosB from S. aureus (FosBSa) and Bacillus cereus (FosBBc). In addition, we have determined a crystal structure of FosBBc with PPF bound in the active site. Our results will be useful for future structure-based development of FosB inhibitors that can be delivered in combination with fosfomycin in order to increase the efficacy of this antibiotic.
Assuntos
Fosfomicina , Antibacterianos/química , Foscarnet/metabolismo , Foscarnet/farmacologia , Fosfomicina/química , Testes de Sensibilidade Microbiana , Staphylococcus aureus/metabolismo , Transferases/metabolismo , Farmacorresistência Bacteriana , Proteínas de Bactérias/metabolismoRESUMO
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. Here, we present a new conformation of 5-LOX where Hα2 adopts an elongated conformation equivalent to that described in other animal lipoxygenase structures. Our observation of the sigmoidal kinetic behavior of 5-LOX, which is indicative of positive cooperativity, is consistent with a substrate-induced conformational change that shifts the ensemble of enzyme populations to favor the catalytically competent state. Strategic point mutations along Hα2 designed to unlock the closed conformation and elongate Hα2 resulted in improved kinetic parameters, altered limited proteolysis data, and a drastic reduction in the length of the lag phase yielding the most active Stable-5-LOX to date. Structural predictions by AlphaFold2 of these variants statistically favor an elongated Hα2 and reinforce a model in which improved kinetic parameters correlate with a more readily adopted open conformation. Taken together, these data provide valuable insights into the synthesis of leukotrienes.
Assuntos
Araquidonato 5-Lipoxigenase , Leucotrienos , Animais , Araquidonato 5-Lipoxigenase/química , Araquidonato 5-Lipoxigenase/genética , Humanos , Ferro/química , Cinética , Leucotrienos/biossíntese , Modelos Moleculares , Mutação Puntual , Conformação Proteica em alfa-Hélice , SolventesRESUMO
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 A4 (LTA4). 5-LOX must traffic to the nuclear membrane to interact with its partner and undergo a conformational change so that AA can enter the active site. Additionally, the enzyme must retain the hydroperoxy-reaction intermediate for its final transformation to LTA4. Each of these steps provide a unique target for inhibition. Next, we describe the recent structures of GPCRs that recognize metabolites of the 5-LOX pathway and thus provide target alternatives. We also highlight the role of 5-LOX in the biosynthesis of anti-inflammatory lipid mediators (LM), the so-called specialized pro-resolving mediators (SPM). The involvement of 5-LOX in the biosynthesis of LM with opposing functions undoubtedly complicates the continuing search for 5-LOX inhibitors as therapeutic leads. Finally, we address the recent discovery of how some allosteric 5-LOX inhibitors promote oxygenation at the 12/15 carbon on AA to generate mediators that resolve, rather than promote, inflammation.
Assuntos
Anti-Inflamatórios/farmacologia , Araquidonato 5-Lipoxigenase/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Lipídeos/farmacologia , Animais , Anti-Inflamatórios/química , Araquidonato 5-Lipoxigenase/genética , Regulação Enzimológica da Expressão Gênica , Humanos , Metabolismo dos Lipídeos , Lipídeos/químicaRESUMO
The pentacyclic triterpenoid quinone methide celastrol (CS) from Tripterygium wilfordii Hook. F. effectively ameliorates inflammation with potential as therapeutics for inflammatory diseases. However, the molecular mechanisms underlying the anti-inflammatory and inflammation-resolving features of CS are incompletely understood. Here we demonstrate that CS potently inhibits the activity of human 5-lipoxygenase (5-LOX), the key enzyme in pro-inflammatory leukotriene (LT) formation, in cell-free assays with IC50 = 0.19-0.49 µM. Employing metabololipidomics using ultra-performance liquid chromatography coupled to tandem mass spectrometry in activated human polymorphonuclear leukocytes or M1 macrophages we found that CS (1 µM) potently suppresses 5-LOX-derived products without impairing the formation of lipid mediators (LM) formed by 12-/15-LOXs as well as fatty acid substrate release. Intriguingly, CS induced the generation of 12-/15-LOX-derived LM including the specialized pro-resolving mediator (SPM) resolvin D5 in human M2 macrophages. Finally, intraperitoneal pre-treatment of mice with 10 mg/kg CS strongly impaired zymosan-induced LT formation and simultaneously elevated the levels of SPM and related 12-/15-LOX-derived LM in peritoneal exudates, spleen and plasma in vivo. Conclusively, CS promotes a switch from LT biosynthesis to formation of SPM which may underlie the anti-inflammatory and inflammation-resolving effects of CS, representing an interesting pharmacological strategy for intervention with inflammatory disorders.
Assuntos
Anti-Inflamatórios/farmacologia , Leucotrienos/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Inibidores de Lipoxigenase/farmacologia , Triterpenos Pentacíclicos/farmacologia , Animais , Anti-Inflamatórios/química , Araquidonato 5-Lipoxigenase/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Células Cultivadas , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inibidores de Lipoxigenase/química , Masculino , Camundongos , Simulação de Acoplamento Molecular , Triterpenos Pentacíclicos/química , Tripterygium/químicaRESUMO
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. The overall structure and the metal ion environments are extremely similar to those of Fe(III)- and Ti(IV)-containing transferrin crystallized under comparable conditions. The octahedral coordination about the Cr(III) is comprised of four ligands provided by the protein (two tyrosine residues, a histidine residue, and an aspartate residue) and a chelating malonate anion. This represents the first crystal structure of a Cr(III)-containing protein that binds Cr(III) as part of its physiological function.
Assuntos
Cromo/metabolismo , Transferrina/metabolismo , Sítios de Ligação , Cromo/química , Cristalografia por Raios X , Humanos , Ligação Proteica , Transferrina/químicaRESUMO
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. The redox-type inhibitor nordihydroguaiaretic acid (NDGA) is lodged in the 5-LOX active site, now fully exposed by disordering of the helix that caps it in the apo-enzyme. In contrast, the allosteric inhibitor 3-acetyl-11-keto-beta-boswellic acid (AKBA) from frankincense wedges between the membrane-binding and catalytic domains of 5-LOX, some 30 Å from the catalytic iron. While enzyme inhibition by NDGA is robust, AKBA promotes a shift in the regiospecificity, evident in human embryonic kidney 293 cells and in primary immune cells expressing 5-LOX. Our results suggest a new approach to isoform-specific 5-LOX inhibitor development through exploitation of an allosteric site in 5-LOX.
Assuntos
Araquidonato 5-Lipoxigenase/química , Produtos Biológicos/química , Inibidores de Lipoxigenase/química , Masoprocol/química , Triterpenos/química , Sítio Alostérico , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/metabolismo , Produtos Biológicos/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Ácidos Hidroxieicosatetraenoicos/química , Ácidos Hidroxieicosatetraenoicos/metabolismo , Leucotrieno B4/química , Leucotrieno B4/metabolismo , Inibidores de Lipoxigenase/metabolismo , Masoprocol/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Triterpenos/metabolismoRESUMO
Mycobacterium abscessus 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 M. abscessus subsp. bolletii that shares approximately 30-55% sequence identity with other vicinal oxygen chelate (VOC) fosfomycin resistance enzymes and represents the first of its type found in any Mycobacterium species. Genes encoding VOC fosfomycin resistance enzymes have been found in both Gram-positive and Gram-negative pathogens. Given that FosA enzymes from Gram-negative bacteria have evolved optimum activity towards glutathione (GSH) and FosB enzymes from Gram-positive bacteria have evolved optimum activity towards bacillithiol (BSH), it was originally suggested that FosM might represent a fourth class of enzyme that has evolved to utilize mycothiol (MSH). However, a sequence similarity network (SSN) analysis identifies FosM as a member of the FosX subfamily, indicating that it may utilize water as a substrate. Here we have synthesized MSH and characterized FosM with respect to divalent metal ion activation and nucleophile selectivity. Our results indicate that FosM is a Mn2+-dependent FosX-type hydrase with no selectivity toward MSH or other thiols as analyzed by NMR and mass spectroscopy.
RESUMO
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.
Assuntos
Antozoários/metabolismo , Araquidonato Lipoxigenases/isolamento & purificação , Ácido Araquidônico/metabolismo , Ensaios Enzimáticos/métodos , Domínios Proteicos/genética , Animais , Araquidonato Lipoxigenases/química , Araquidonato Lipoxigenases/genética , Araquidonato Lipoxigenases/metabolismo , Ácido Araquidônico/química , Sítios de Ligação/genética , Cristalografia por Raios X/métodos , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato/genéticaRESUMO
A unique aspect of arrestin-3 is its ability to support both receptor-dependent and receptor-independent signaling. Here, we show that inositol hexakisphosphate (IP6) is a non-receptor activator of arrestin-3 and report the structure of IP6-activated arrestin-3 at 2.4-Å resolution. IP6-activated arrestin-3 exhibits an inter-domain twist and a displaced C-tail, hallmarks of active arrestin. IP6 binds to the arrestin phosphate sensor, and is stabilized by trimerization. Analysis of the trimerization surface, which is also the receptor-binding surface, suggests a feature called the finger loop as a key region of the activation sensor. We show that finger loop helicity and flexibility may underlie coupling to hundreds of diverse receptors and also promote arrestin-3 activation by IP6. Importantly, we show that effector-binding sites on arrestins have distinct conformations in the basal and activated states, acting as switch regions. These switch regions may work with the inter-domain twist to initiate and direct arrestin-mediated signaling.
Assuntos
Arrestinas/química , Arrestinas/metabolismo , Sequência de Aminoácidos , Animais , Arrestinas/genética , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Humanos , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Ácido Fítico/metabolismo , Conformação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
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 2.47-Å resolution crystal structure of the arachidonate 11R-LOX from Gersemia fruticosa, which sheds new light on the mechanism of LOX catalysis. Our crystallographic and mutational studies suggest that the aliphatic tail of the fatty acid is bound in a hydrophobic pocket with two potential entrances. We speculate that LOXs share a common T-shaped substrate channel architecture that gives rise to the varying positional specificities. A general allosteric mechanism is proposed for transmitting the activity-inducing effect of calcium binding from the membrane-targeting PLAT (polycystin-1/lipoxygenase/α-toxin) domain to the active site via a conserved π-cation bridge.
Assuntos
Araquidonato Lipoxigenases/química , Cálcio/metabolismo , Regulação da Expressão Gênica , Sítio Alostérico , Animais , Antozoários , Proteínas de Ligação ao Cálcio/metabolismo , Domínio Catalítico , Membrana Celular/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Cristalografia por Raios X/métodos , Dimerização , Eicosanoides/química , Humanos , Cinética , Lipossomos/metabolismo , Espectrometria de Massas/métodos , Modelos Químicos , Conformação Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de ProteínaRESUMO
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 S663D enzyme exhibits robust 15-LOX activity, as determined by spectrophotometric and HPLC analyses, with only traces of 5-LOX activity remaining; synthesis of the anti-inflammatory lipoxin A(4) from arachidonic acid is also detected. The crystal structure of the S663D mutant in the absence and presence of arachidonic acid (in the context of the previously reported Stable-5-LOX) reveals substantial remodeling of helices that define the active site so that the once fully encapsulated catalytic machinery is solvent accessible. Our results suggest that phosphorylation of 5-LOX at S663 could not only down-regulate leukotriene synthesis but also stimulate lipoxin production in inflammatory cells that do not express 15-LOX, thus redirecting lipid mediator biosynthesis to the production of proresolving mediators of inflammation.
Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Humanos , Lipoxinas/biossíntese , Modelos Moleculares , Fosforilação , Mutação Puntual , Serina/metabolismoRESUMO
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.
Assuntos
Araquidonato 5-Lipoxigenase/química , Sequência de Aminoácidos , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Estabilidade Enzimática , Humanos , Ferro/química , Ferro/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
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. E., Hansson, G., Hedqvist, P., Björck, T., Granström, E., and Dahlén, B. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1712-1716). Over the last 25+ years, substantial progress has been made in understanding how LTs exert their effects, and a broader appreciation for the numerous biological processes they mediate has emerged. LT biosynthesis is initiated by the action of 5-lipoxygenase (5-LOX), which catalyzes the transformation of AA to LTA(4) in a two-step reaction. Ca(2+) targets 5-LOX to the nuclear membrane, where it co-localizes with the 5-LOX-activating protein FLAP and, when present, the downstream enzyme LTC(4) synthase, both transmembrane proteins. Crystal structures of the AA-metabolizing LOXs, LTC(4) synthase, and FLAP combined with biochemical data provide a framework for understanding how subcellular organizations optimize the biosynthesis of these labile hydrophobic signaling compounds, which must navigate pathways that include both membrane and soluble enzymes. The insights these structures afford and the questions they engender are discussed in this minireview.