RESUMEN
Glycoside hydrolases (GHs) are involved in the degradation of a wide diversity of carbohydrates and present several biotechnological applications. Many GH families are composed of enzymes with a single well-defined specificity. In contrast, enzymes from the GH16 family can act on a range of different polysaccharides, including ß-glucans and galactans. SCLam, a GH16 member derived from a soil metagenome, an endo-ß-1,3(4)-glucanase (EC 3.2.1.6), can cleave both ß-1,3 and ß-1,4 glycosidic bonds in glucans, such as laminarin, barley ß-glucan, and cello-oligosaccharides. A similar cleavage pattern was previously reported for other GH16 family members. However, the molecular mechanisms for this dual cleavage activity on (1,3)- and (1,4)-ß-D-glycosidic bonds by laminarinases have not been elucidated. In this sense, we determined the X-ray structure of a presumably inactive form of SCLam cocrystallized with different oligosaccharides. The solved structures revealed general bound products that are formed owing to residual activities of hydrolysis and transglycosylation. Biochemical and biophysical analyses and molecular dynamics simulations help to rationalize differences in activity toward different substrates. Our results depicted a bulky aromatic residue near the catalytic site critical to select the preferable configuration of glycosidic bonds in the binding cleft. Altogether, these data contribute to understanding the structural basis of recognition and hydrolysis of ß-1,3 and ß-1,4 glycosidic linkages of the laminarinase enzyme class, which is valuable for future studies on the GH16 family members and applications related to biomass conversion into feedstocks and bioproducts.
Asunto(s)
Proteínas Bacterianas/metabolismo , Celulasas/metabolismo , Glucanos/metabolismo , Proteínas Bacterianas/química , Secuencia de Carbohidratos , Dominio Catalítico , Celulasas/química , Cristalografía por Rayos X/métodos , Glucanos/clasificación , Glicósidos/química , Glicósidos/metabolismo , Hidrólisis , Simulación de Dinámica Molecular , Microbiología del Suelo , Especificidad por SustratoRESUMEN
Knowing the key features of the structure and the biochemistry of proteins is crucial to improving enzymes of industrial interest like ß-fructofuranosidase. Gene sacA from Bacillus licheniformis ATCC 14580 codifies a sucrose-6-phosphate hydrolase, a ß-fructofuranosidase (E.C. 3.1.2.26, protein BlsacA), which has no crystallographic structure available. In this study, we report the results from numerous biochemical and biophysical techniques applied to the investigation of BlsacA in solution. BlsacA was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Results showed that the optimum activity of BlsacA occurred at 30 °C around neutrality (pH 6.0-7.5) with a tendency to alkalinity. Circular dichroism spectrum confirmed that BlsacA contains elements of a ß-sheet secondary structure at the optimum pH range and the maintenance of these elements is related to BlsacA enzymatic stability. Dynamic light scattering and small-angle X-ray scattering measurements showed that BlsacA forms stable and elongated homodimers which displays negligible flexibility in solution at optimum pH range. The BlsacA homodimeric nature is strictly related to its optimum activity and is responsible for the generation of biphasic curves during differential scanning fluorimetry analyses. The homodimer is formed through the contact of the N-terminal ß-propeller domain of each BlsacA unit. The results presented here resemble the key importance of the homodimeric form of BlsacA for the enzyme stability and the optimum enzymatic activity.
Asunto(s)
Bacillus licheniformis/enzimología , Sacarosa/análogos & derivados , Fosfatos de Azúcar/metabolismo , beta-Fructofuranosidasa/química , beta-Fructofuranosidasa/metabolismo , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Estructura Secundaria de Proteína , Dispersión del Ángulo Pequeño , Especificidad por Sustrato , Sacarosa/metabolismo , Difracción de Rayos XRESUMEN
Peroxisome Proliferator-Activated Receptors (PPARs) are ligand-dependent transcription factors that control various functions in human organism, including the control of glucose and lipid metabolism. PPARγ is a target of TZD agonists, clinically used to improve insulin sensitivity whereas fibrates, PPARα ligands, lower serum triglyceride levels. We report here the structural studies of GL479, a synthetic dual PPARα/γ agonist, designed by a combination of clofibric acid skeleton and a phenyldiazenyl moiety, as bioisosteric replacement of stilbene group, in complex with both PPARα and PPARγ receptors. GL479 was previously reported as a partial agonist of PPARγ and a full agonist of PPARα with high affinity for both PPARs. Our structural studies reveal different binding modes of GL479 to PPARα and PPARγ, which may explain the distinct activation behaviors observed for each receptor. In both cases the ligand interacts with a Tyr located at helix 12 (H12), resulting in the receptor active conformation. In the complex with PPARα, GL479 occupies the same region of the ligand-binding pocket (LBP) observed for other full agonists, whereas GL479 bound to PPARγ displays a new binding mode. Our results indicate a novel region of PPARs LBP that may be explored for the design of partial agonists as well dual PPARα/γ agonists that combine, simultaneously, the therapeutic effects of the treatment of insulin resistance and dyslipidemia.
Asunto(s)
PPAR alfa/agonistas , PPAR alfa/química , PPAR gamma/agonistas , PPAR gamma/química , Sitios de Unión , Ligandos , Unión Proteica , Estructura Secundaria de Proteína , Tomografía por Rayos X/métodosRESUMEN
Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for ß-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl ß-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Celulasas/genética , Celulasas/metabolismo , Celulosa/metabolismo , Trichoderma/enzimología , Celulasas/química , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Temperatura , Trichoderma/genéticaRESUMEN
The lack of synthesized information regarding biodiversity is a major problem among researchers, leading to a pervasive cycle where ecologists make field campaigns to collect information that already exists and yet has not been made available for a broader audience. This problem leads to long-lasting effects in public policies such as spending money multiple times to conduct similar studies in the same area. We aim to identify this knowledge gap by synthesizing information available regarding two Brazilian long-term biodiversity programs and the metadata generated by them. Using a unique dataset containing 1904 metadata, we identified patterns of metadata distribution and intensity of research conducted in Brazil, as well as where we should concentrate research efforts in the next decades. We found that the majority of metadata were about vertebrates, followed by plants, invertebrates, and fungi. Caatinga was the biome with least metadata, and that there's still a lack of information regarding all biomes in Brazil, with none of them being sufficiently sampled. We hope that these results will have implications for broader conservation and management guiding, as well as to funding allocation programs.
RESUMEN
Xylose isomerase (EC 5.3.1.5) is a key enzyme in xylose metabolism which is industrially important for the transformation of glucose and xylose into fructose and xylulose, respectively. The Bifidobacterium adolescentis xylA gene (NC_008618.1) encoding xylose isomerase (XI) was cloned and the enzyme was overexpressed in Escherichia coli. Purified recombinant XI was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 3350 as the precipitating agent. A complete native data set was collected to 1.7â Å resolution using a synchrotron-radiation source. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 88.78, b = 123.98, c = 78.63â Å.
Asunto(s)
Isomerasas Aldosa-Cetosa/genética , Isomerasas Aldosa-Cetosa/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Bifidobacterium/enzimología , Regulación Bacteriana de la Expresión Génica , Isomerasas Aldosa-Cetosa/química , Proteínas Bacterianas/química , Bifidobacterium/genética , Cristalización , Difracción de Rayos XRESUMEN
The peroxisome proliferator-activated receptor γ (PPARγ) is a target for treatment of type II diabetes and other conditions. PPARγ full agonists, such as thiazolidinediones (TZDs), are effective insulin sensitizers and anti-inflammatory agents, but their use is limited by adverse side effects. Luteolin is a flavonoid with anti-inflammatory actions that binds PPARγ but, unlike TZDs, does not promote adipocyte differentiation. However, previous reports suggested variously that luteolin is a PPARγ agonist or an antagonist. We show that luteolin exhibits weak partial agonist/antagonist activity in transfections, inhibits several PPARγ target genes in 3T3-L1 cells (LPL, ORL1, and CEBPα) and PPARγ-dependent adipogenesis, but activates GLUT4 to a similar degree as rosiglitazone, implying gene-specific partial agonism. The crystal structure of the PPARγ ligand-binding domain (LBD) reveals that luteolin occupies a buried ligand-binding pocket (LBP) but binds an inactive PPARγ LBD conformer and occupies a space near the ß-sheet region far from the activation helix (H12), consistent with partial agonist/antagonist actions. A single myristic acid molecule simultaneously binds the LBP, suggesting that luteolin may cooperate with other ligands to bind PPARγ, and molecular dynamics simulations show that luteolin and myristic acid cooperate to stabilize the Ω-loop among H2', H3, and the ß-sheet region. It is noteworthy that luteolin strongly suppresses hypertonicity-induced release of the pro-inflammatory interleukin-8 from human corneal epithelial cells and reverses reductions in transepithelial electrical resistance. This effect is PPARγ-dependent. We propose that activities of luteolin are related to its singular binding mode, that anti-inflammatory activity does not require H12 stabilization, and that our structure can be useful in developing safe selective PPARγ modulators.
Asunto(s)
Luteolina/farmacología , PPAR gamma/agonistas , Células 3T3-L1 , Animales , Secuencia de Bases , Cartilla de ADN , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Luteolina/química , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , Ácido Mirístico/química , PPAR gamma/química , PPAR gamma/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rosiglitazona , Tiazolidinedionas/antagonistas & inhibidores , Tiazolidinedionas/farmacologíaRESUMEN
Fructooligosaccharides (FOSs) are polymers of fructose with a prebiotic activity because of their production and fermentation by bacteria that inhabit the gastrointestinal tract and are widely used in the industry and new functional foods. Lactobacillus gasseri stands out as an important homofermentative microorganism related to FOS production, and its potential applications in the industry are undeniable. In this study, we report the production and characterization of a sucrose-6-phosphate hydrolase from L. gasseri belonging to the GH32 family. Apo-LgAs32 and LgAs32 complexed with ß-d-fructose structures were determined at a resolution of 1.94 and 1.84 Å, respectively. The production of FOS, fructans, 1-kestose, and nystose by the recombinant LgAs32, using sucrose as a substrate, shown in this study is very promising. When compared to its homologous enzyme from Lactobacillus reuteri, the production of 1-kestose by LgAs32 is increased; thus, LgAs32 can be considered as an alternative in fructan production and other industrial applications.
Asunto(s)
Hexosiltransferasas , Lactobacillus gasseri , Industria de Alimentos , Fructanos , Hexosiltransferasas/genética , Oligosacáridos , Sacarosa/análogos & derivados , Fosfatos de Azúcar , beta-Fructofuranosidasa/genéticaRESUMEN
The fungus Thermothielavioides terrestris plays an important role in the global carbon cycle with enzymes capable of degrading polysaccharides from biomass, therefore an attractive source of proteins to be investigated and understood. From cloning to a three-dimensional structure, we foster a deeper characterization of an α-Ê-arabinofuranosidase, a glycoside hydrolase from the family 62 (TtAbf62), responsible to release arabinofuranose from non-reducing ends of polysaccharides. TtAbf62 was tested with synthetic (pNP-Araf) and polymeric substrates (arabinan and arabinoxylan), showing optimal temperature and pH (for pNP-Araf) of 30 °C and 4.5-5.0, respectively. Kinetic parameters revealed different specific activity for the three substrates, with a higher affinity for pNP-Araf (KM: 4 ± 1 mM). The hydrolyzing activity of TtAbf62 on sugarcane bagasse suggests high efficiency in the decomposition of arabinoxylan, abundant hemicellulose presented in the sugarcane cell wall. The crystal packing of TtAbf62 reveals an exquisite domain swapping, located at the supramolecular arrangement through a disulfide bond. All crystallographic behaviors go against its monomeric state in solution, indicating a crystal-induced artifact. Structural information will form the basis for further studies aiming the development of optimized enzymatic properties to be used in biotechnological applications.
Asunto(s)
Ascomicetos/enzimología , Glicósido Hidrolasas/química , Modelos Moleculares , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Fenómenos Bioquímicos , Catálisis , Glicósido Hidrolasas/metabolismo , Cinética , Unión Proteica , Proteínas Recombinantes , Análisis Espectral , Relación Estructura-ActividadRESUMEN
BACKGROUND: Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron transfer between NADP(H) and the proteins ferredoxin or flavodoxin. A number of structural features distinguish plant and bacterial FNRs, one of which is the mode of the cofactor FAD binding. Leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. Leptospira interrogans FNR (LepFNR) displays low sequence identity with plant (34% with Zea mays) and bacterial (31% with Escherichia coli) FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs. RESULTS: The crystal structures of the FAD-containing LepFNR and the complex of the enzyme with NADP+, were solved and compared to known FNRs. The comparison reveals significant structural similarities of the enzyme with the plastidic type FNRs and differences with the bacterial enzymes. Our small angle X-ray scattering experiments show that LepFNR is a monomeric enzyme. Moreover, our biochemical data demonstrate that the LepFNR has an enzymatic activity similar to those reported for the plastidic enzymes and that is significantly different from bacterial flavoenzymes, which display lower turnover rates. CONCLUSION: LepFNR is the first plastidic type FNR found in bacteria and, despite of its low sequence similarity with plastidic FNRs still displays high catalytic turnover rates. The typical structural and biochemical characteristics of plant FNRs unveiled for LepFNR support a notion of a putative lateral gene transfer which presumably offers Leptospira interrogans evolutionary advantages. The wealth of structural information about LepFNR provides a molecular basis for advanced drugs developments against leptospirosis.
Asunto(s)
Ferredoxina-NADP Reductasa/química , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/metabolismo , Leptospira interrogans/enzimología , NADP/metabolismo , Secuencia de Aminoácidos , Ácido Aspártico/química , Bacterias/enzimología , Sitios de Unión , Cristalografía por Rayos X , Escherichia coli/genética , Ferredoxina-NADP Reductasa/aislamiento & purificación , Ferredoxina-NADP Reductasa/metabolismo , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Plantas/enzimología , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Homología de Secuencia de Aminoácido , Temperatura , Difracción de Rayos XRESUMEN
OBJECTIVE: To compare DMARD use in patients with and without FM over time, including overtreatment and undertreatment rates in both groups. METHODS: A prospective cohort study with patients attending an RA outpatient clinic was conducted. Participants were consecutively recruited between March 2006 and June 2007 and were followed through December 2013. Data on DMARD use (prevalences, doses and escalation rates), DAS28, HAQ and radiographic progression were compared among RA patients with FM and without FM. Mistreatment clinical scenarios were allegedly identified and compared between groups. RESULTS: 256 RA patients (32 with FM) were followed for 6.2±2.0 (mean±SD) years comprising 2986 visits. At baseline, RA duration was 11.1±7.4 years. DAS28 and HAQ were greater in RA with FM group, and were closer to RA without FM group towards the end. RA patients with FM used higher doses of tricyclic antidepressants, leflunomide and prednisone, and lower doses of methotrexate. When compared to RA patients without FM, participants with RA and FM used more often tricyclic antidepressants, leflunomide, prednisone, continuous analgesics and less often methotrexate. Groups presented similar 7-year biologic-free survival, and radiographic progression-free survival in Cox regression. RA patients with FM had greater proportions of visits in mistreatment scenarios when compared to RA patients without FM (28.4 vs. 19.8%, p<0.001). CONCLUSIONS: RA patients with FM used more leflunomide and prednisone, and RA mistreatment was more frequent in FM patients. Certainly, RA patients with FM will benefit from a personalized T2T strategy, including ultrasound (when suitable) and proper FM treatment.
Asunto(s)
Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Toma de Decisiones Clínicas , Fibromialgia/complicaciones , Prescripción Inadecuada/estadística & datos numéricos , Pautas de la Práctica en Medicina/estadística & datos numéricos , Adulto , Anciano , Artritis Reumatoide/complicaciones , Brasil , Estudios de Casos y Controles , Progresión de la Enfermedad , Esquema de Medicación , Femenino , Estudios de Seguimiento , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Índice de Severidad de la EnfermedadRESUMEN
OBJECTIVE: To compare DMARD use in patients with and without FM over time, including overtreatment and undertreatment rates in both groups. METHODS: A prospective cohort study with patients attending an RA outpatient clinic was conducted. Participants were consecutively recruited between March 2006 and June 2007 and were followed through December 2013. Data on DMARD use (prevalences, doses and escalation rates), DAS28, HAQ and radiographic progression were compared among RA patients with FM and without FM. Mistreatment clinical scenarios were allegedly identified and compared between groups. RESULTS: 256 RA patients (32 with FM) were followed for 6.2±2.0 (mean±SD) years comprising 2,986 visits. At baseline, RA duration was 11.1±7.4 years. DAS28 and HAQ were greater in RA with FM group, and were closer to RA without FM group towards the end. RA patients with FM used higher doses of tricyclic antidepressants, leflunomide and prednisone, and lower doses of methotrexate. When compared to RA patients without FM, participants with RA and FM used more often tricyclic antidepressants, leflunomide, prednisone, continuous analgesics and less often methotrexate. Groups presented similar 7-year biologic-free survival, and radiographic progression-free survival in Cox regression. RA patients with FM had greater proportions of visits in mistreatment scenarios when compared to RA patients without FM (28.4 vs. 19.8%, p<0.001). CONCLUSIONS: RA patients with FM used more leflunomide and prednisone, and RA mistreatment was more frequent in FM patients. Certainly, RA patients with FM will benefit from a personalized T2T strategy, including ultrasound (when suitable) and proper FM treatment.
RESUMEN
Palm tree peroxidases are known to be very stable enzymes and the peroxidase from the Chamaerops excelsa (CEP), which has a high pH and thermal stability, is no exception. To date, the structural and molecular events underscoring such biochemical behavior have not been explored in depth. In order to identify the structural characteristics accounting for the high stability of palm tree peroxidases, we solved and refined the X-ray structure of native CEP at a resolution of 2.6 Å. The CEP structure has an overall fold typical of plant peroxidases and confirmed the conservation of characteristic structural elements such as the heme group and calcium ions. At the same time the structure revealed important modifications in the amino acid residues in the vicinity of the exposed heme edge region, involved in substrate binding, that could account for the morphological variations among palm tree peroxidases through the disruption of molecular interactions at the second binding site. These modifications could alleviate the inhibition of enzymatic activity caused by molecular interactions at the latter binding site. Comparing the CEP crystallographic model described here with other publicly available peroxidase structures allowed the identification of a noncovalent homodimer assembly held together by a number of ionic and hydrophobic interactions. We demonstrate, that this dimeric arrangement results in a more stable protein quaternary structure through stabilization of the regions that are highly dynamic in other peroxidases. In addition, we resolved five N-glycosylation sites, which might also contribute to enzyme stability and resistance against proteolytic cleavage.
Asunto(s)
Arecaceae/enzimología , Peroxidasa/química , Proteínas de Plantas/química , Cristalografía por Rayos X , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Non-steroidal anti-inflammatory drugs (NSAIDs) display anti-inflammatory, antipyretic and analgesic properties by inhibiting cyclooxygenases and blocking prostaglandin production. Previous studies, however, suggested that some NSAIDs also modulate peroxisome proliferator activated receptors (PPARs), raising the possibility that such off target effects contribute to the spectrum of clinically relevant NSAID actions. In this study, we set out to understand how peroxisome proliferator activated receptor-γ (PPARγ/PPARG) interacts with NSAIDs using X-ray crystallography and to relate ligand binding modes to effects on receptor activity. We find that several NSAIDs (sulindac sulfide, diclofenac, indomethacin and ibuprofen) bind PPARγ and modulate PPARγ activity at pharmacologically relevant concentrations. Diclofenac acts as a partial agonist and binds to the PPARγ ligand binding pocket (LBP) in typical partial agonist mode, near the ß-sheets and helix 3. By contrast, two copies of indomethacin and sulindac sulfide bind the LBP and, in aggregate, these ligands engage in LBP contacts that resemble agonists. Accordingly, both compounds, and ibuprofen, act as strong partial agonists. Assessment of NSAID activities in PPARγ-dependent 3T3-L1 cells reveals that NSAIDs display adipogenic activities and exclusively regulate PPARγ-dependent target genes in a manner that is consistent with their observed binding modes. Further, PPARγ knockdown eliminates indomethacin activities at selected endogenous genes, confirming receptor-dependence of observed effects. We propose that it is important to consider how individual NSAIDs interact with PPARγ to understand their activities, and that it will be interesting to determine whether high dose NSAID therapies result in PPAR activation.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , PPAR gamma/metabolismo , Células 3T3-L1 , Adipogénesis/efectos de los fármacos , Animales , Agonismo Parcial de Drogas , Células HeLa , Humanos , Ratones , Modelos Moleculares , PPAR gamma/agonistas , PPAR gamma/química , Conformación ProteicaRESUMEN
Glycoside hydrolases (GHs) are enzymes found in all living kingdoms that are involved in multiple physiological functions. Due to their multiple enzymatic activities, GHs are broadly applied in bioethanol, food, and paper industry. In order to increase the productivity of these industrial processes, a constant search for novel and efficient enzymes has been proved to be necessary. In this context, metagenomics is a powerful approach to achieve this demand. In the current study, we describe the discovery and characterization of a novel member of GH16 family derived from the sugarcane soil metagenome. The enzyme, named SCLam, has 286 amino acid residues and displays sequence homology and activity properties that resemble known laminarases. SCLam is active against barley beta-glucan, laminarin, and lichenan (72, 33, and 10 U mg(-1), respectively). The optimal reaction conditions were identified as 40 °C and pH 6.5. The low-resolution structure was determined using the small-angle X-ray scattering technique, revealing that SCLam is a monomer in solution with a radius of gyration equal to 19.6 Å. To the best of our knowledge, SCLam is the first nonspecific (1,3/1,3:1,4)-ß-D-glucan endohydrolase (EC 3.2.1.6) recovered by metagenomic approach to be characterized.
Asunto(s)
Glicósido Hidrolasas/metabolismo , Metagenoma , Saccharum/crecimiento & desarrollo , Microbiología del Suelo , Secuencia de Aminoácidos , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Hidrólisis , Filogenia , Dispersión del Ángulo Pequeño , Especificidad por Sustrato , Temperatura , Difracción de Rayos XRESUMEN
The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-ß-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on ß-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50 °C on ß-glucan. Under these conditions specific activity was 239.2 ± 9.1 U mg(-1) and the half-life of the enzyme was 84.6 ± 3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using ß-glucan as a substrate, the Km was 3.2 ± 0.5 mg mL(-1) and the Vmax was 0.41 ± 0.02 µmol min(-1). Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.
Asunto(s)
Basidiomycota/enzimología , Celulasa/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , beta-Glucanos/metabolismo , Aspergillus niger/genética , Basidiomycota/genética , Celulasa/genética , Clonación Molecular/métodos , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Espectrometría de Masas/métodos , Microscopía Electrónica de Rastreo/métodos , Proteínas Recombinantes/aislamiento & purificación , Especificidad por Sustrato , TemperaturaRESUMEN
Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear transcription factors. They are involved in mediating numerous physiological effects in humans, including glucose and lipid metabolism. PPARα ligands effectively treat dyslipidemia and have significant antiinflammatory and anti-atherosclerotic activities. These effects and their ligand-dependent activity make nuclear receptors obvious targets for drug design. Here, we present the structure of the human PPARα in complex with WY14643, a member of fibrate class of drug, and a widely used PPAR activator. The crystal structure of this complex suggests that WY14643 induces activation of PPARα in an unusual bipartite mechanism involving conventional direct helix 12 stabilization and an alternative mode that involves a second ligand in the pocket. We present structural observations, molecular dynamics and activity assays that support the importance of the second site in WY14643 action. The unique binding mode of WY14643 reveals a new pattern of nuclear receptor ligand recognition and suggests a novel basis for ligand design, offering clues for improving the binding affinity and selectivity of ligand. We show that binding of WY14643 to PPARα was associated with antiinflammatory disease in a human corneal cell model, suggesting possible applications for PPARα ligands.
Asunto(s)
PPAR alfa/agonistas , PPAR alfa/química , Pirimidinas/química , Pirimidinas/metabolismo , Antiinflamatorios/química , Antiinflamatorios/metabolismo , Células Cultivadas , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Cinética , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
A manutenção de uma auto imagem positiva tem relação direta a dentição do indivíduo. A utilização de próteses sejam elas parciais ou totais, tem papel fundamental tanto na saúde oral, quanto na saúde sistêmica do paciente. O restabelecimento da função oclusal, a preservação de estruturas orais remanescentes dependem diretamente de um planejamento protético rigoroso. A satisfação do paciente é fator determinante do sucesso ou insucesso de uma reabilitação protética, bem como o restabelecimento do bem-estar físico, mental e social. Paciente sexo feminino, 32 anos, relata insatisfação estética 120 dias após reabilitação estética com prótese parcial removível. A oroscopia observou-se, inclinação para distal dos elementos 12 e 21 bem como excesso de material restaurador. Observou-se ainda lesão séssil de cor avermelhada em gengiva inserida na região do elemento 11 (ausente), bordas delimitadas, apresentando dor provocada ao toque, sem sangramento e de surgimento a aproximadamente 12 meses, sendo anterior a instalação da PPR. Realizado biópsia excisional, com histopatologia compatível ao granuloma. Tratamento endodôntico do elemento 21. Remoção de carie dos elementos acometidos, inclusive pilar protético. Faceta direta em Resina Fotopolimerizável nos elementos 12 e 21. Recuperação da estética dentaria e saúde dos tecidos de suporte. O objetivo do presente está em destacar aspectos relacionados à falhas de PPR se não corretamente diagnosticadas, avaliadas e prevenidas.(AU)
Asunto(s)
Humanos , Femenino , Adulto , Fracaso de la Restauración Dental , Dentadura Parcial Removible/efectos adversos , Estética Dental , Radiografía Dental , Resultado del Tratamiento , Granuloma Piogénico/patología , Enfermedades de las Encías/patologíaRESUMEN
The peroxisome proliferator-activated receptors (PPARs) regulate genes involved in lipid and carbohydrate metabolism, and are targets of drugs approved for human use. Whereas the crystallographic structure of the complex of full length PPARγ and RXRα is known, structural alterations induced by heterodimer formation and DNA contacts are not well understood. Herein, we report a small-angle X-ray scattering analysis of the oligomeric state of hPPARγ alone and in the presence of retinoid X receptor (RXR). The results reveal that, in contrast with other studied nuclear receptors, which predominantly form dimers in solution, hPPARγ remains in the monomeric form by itself but forms heterodimers with hRXRα. The low-resolution models of hPPARγ/RXRα complexes predict significant changes in opening angle between heterodimerization partners (LBD) and extended and asymmetric shape of the dimer (LBD-DBD) as compared with X-ray structure of the full-length receptor bound to DNA. These differences between our SAXS models and the high-resolution crystallographic structure might suggest that there are different conformations of functional heterodimer complex in solution. Accordingly, hydrogen/deuterium exchange experiments reveal that the heterodimer binding to DNA promotes more compact and less solvent-accessible conformation of the receptor complex.
Asunto(s)
Modelos Moleculares , PPAR gamma/química , Secuencia de Aminoácidos , Cromatografía en Gel , ADN/metabolismo , Medición de Intercambio de Deuterio , Humanos , Hidrodinámica , Espectrometría de Masas , Datos de Secuencia Molecular , PPAR gamma/metabolismo , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Elementos de Respuesta/genética , Receptor alfa X Retinoide/química , Receptor alfa X Retinoide/metabolismo , Dispersión del Ángulo Pequeño , Soluciones , Difracción de Rayos XRESUMEN
Peroxisome proliferator activated receptors (PPARs δ, α and γ) are closely related transcription factors that exert distinct effects on fatty acid and glucose metabolism, cardiac disease, inflammatory response and other processes. Several groups developed PPAR subtype specific modulators to trigger desirable effects of particular PPARs without harmful side effects associated with activation of other subtypes. Presently, however, many compounds that bind to one of the PPARs cross-react with others and rational strategies to obtain highly selective PPAR modulators are far from clear. GW0742 is a synthetic ligand that binds PPARδ more than 300-fold more tightly than PPARα or PPARγ but the structural basis of PPARδ:GW0742 interactions and reasons for strong selectivity are not clear. Here we report the crystal structure of the PPARδ:GW0742 complex. Comparisons of the PPARδ:GW0742 complex with published structures of PPARs in complex with α and γ selective agonists and pan agonists suggests that two residues (Val312 and Ile328) in the buried hormone binding pocket play special roles in PPARδ selective binding and experimental and computational analysis of effects of mutations in these residues confirms this and suggests that bulky substituents that line the PPARα and γ ligand binding pockets as structural barriers for GW0742 binding. This analysis suggests general strategies for selective PPARδ ligand design.