RESUMEN

The escalating prevalence of drug-resistant pathogens not only jeopardizes the effectiveness of existing treatments but also increases the complexity and severity of infectious diseases. Escherichia coli is one the most common pathogens across all healthcare-associated infections. Enzymatic treatment of bacterial biofilms, targeting extracellular polymeric substances (EPS), can be used for EPS degradation and consequent increase in susceptibility of pathogenic bacteria to antibiotics. Here, we characterized three recombinant cellulases from Thermothelomyces thermophilus: a cellobiohydrolase I (TthCel7A), an endoglucanase (TthCel7B), and a cellobiohydrolase II (TthCel6A) as tools for hydrolysis of E. coli and Gluconacetobacter hansenii biofilms. Using a design mixture approach, we optimized the composition of cellulases, enhancing their synergistic activity to degrade the biofilms and significantly reducing the enzymatic dosage. In line with the crystalline and ordered structure of bacterial cellulose, the mixture of exo-glucanases (0.5 TthCel7A:0.5 TthCel6A) is effective in the hydrolysis of G. hansenii biofilm. Meanwhile, a mixture of exo- and endo-glucanases is required for the eradication of E. coli 042 and clinical E. coli biofilms with significantly different proportions of the enzymes (0.56 TthCel7B:0.44 TthCel6A and 0.6 TthCel7A:0.4 TthCel7B, respectively). X-ray diffraction pattern and crystallinity index of E. coli cellulose are comparable to those of carboxymethyl cellulose (CMC) substrate. Our results illustrate the complexity of E. coli biofilms and show that successful hydrolysis is achieved by a specific combination of cellulases, with consistent recurrence of TthCel7B endoglucanase.

Asunto(s)

Biopelículas , Escherichia coli , Biopelículas/crecimiento & desarrollo , Biopelículas/efectos de los fármacos , Hidrólisis , Celulasa/metabolismo , Celulasa/química , Hypocreales/enzimología

RESUMEN

Glycoside hydrolase family 5 (GH5) encompasses enzymes with several different activities, including endo-1,4-ß-mannosidases. These enzymes are involved in mannan degradation, and have a number of biotechnological applications, such as mannooligosaccharide prebiotics production, stain removal and dyes decolorization, to name a few. Despite the importance of GH5 enzymes, only a few members of subfamily 7 were structurally characterized. In the present work, biochemical and structural characterization of Bacillus licheniformis GH5 mannanase, BlMan5_7 were performed and the enzyme cleavage pattern was analyzed, showing that BlMan5_7 requires at least 5 occupied subsites to perform efficient hydrolysis. Additionally, crystallographic structure at 1.3 Å resolution was determined and mannoheptaose (M7) was docked into the active site to investigate the interactions between substrate and enzyme through molecular dynamic (MD) simulations, revealing the existence of a - 4 subsite, which might explain the generation of mannotetraose (M4) as an enzyme product. Biotechnological application of the enzyme in stain removal was investigated, demonstrating that BlMan5_7 addition to washing solution greatly improves mannan-based stain elimination.

Asunto(s)

Bacillus licheniformis , Dominio Catalítico , Mutagénesis Sitio-Dirigida , Bacillus licheniformis/enzimología , Bacillus licheniformis/genética , Cristalografía por Rayos X , Simulación de Dinámica Molecular , Manosidasas/química , Manosidasas/genética , Manosidasas/metabolismo , Especificidad por Sustrato , Hidrólisis , Tetrosas/química , Tetrosas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Conformación Proteica , Mananos/química , Mananos/metabolismo , beta-Manosidasa/química , beta-Manosidasa/genética , beta-Manosidasa/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , Oligosacáridos

RESUMEN

Leptospirosis is a bacterial disease that affects humans and animals and is caused by Leptospira. The recommended treatment for leptospirosis is antibiotic therapy, which should be given early in the course of the disease. Despite the use of these antibiotics, their role during the course of the disease is still not completely clear because of the lack of effective clinical trials, particularly for severe cases of the disease. Here, we present the characterization of L. interrogans Lsa45 protein by gel filtration, protein crystallography, SAXS, fluorescence and enzymatic assays. The oligomeric studies revealed that Lsa45 is monomeric in solution. The crystal structure of Lsa45 revealed the presence of two subdomains: a large α/ß subdomain and a small α-helical subdomain. The large subdomain contains the amino acids Ser122, Lys125, and Tyr217, which correspond to the catalytic triad that is essential for ß-lactamase or serine hydrolase activity in similar enzymes. Additionally, we also confirmed the bifunctional promiscuity of Lsa45, in hydrolyzing both the 4-nitrophenyl acetate (p-NPA) and nitrocefin ß-lactam antibiotic. Therefore, this study provides novel insights into the structure and function of enzymes from L. interrogans, which furthers our understanding of this bacterium and the development of new therapies for the prevention and treatment of leptospirosis.

RESUMEN

SAXSMoW (SAXS Molecular Weight) is an online platform widely used over the past few years for determination of molecular weights of proteins in dilute solutions. The scattering intensity retrieved from small-angle X-ray scattering (SAXS) raw data is the sole input to SAXSMoW for determination of molecular weights of proteins in liquid. The current updated SAXSMoW version 3.0 determines the linear dependence of the true protein volume on their apparent protein volume, based on SAXS curves calculated for 67,000 protein structures selected from the Protein Data Bank. SAXSMoW 3.0 was tested against 43 experimental SAXS scattering curves from proteins with known molecular weights. Our results demonstrate that most of the molecular weights determined for the nonglycosylated and also for the glycosylated proteins are in good agreement with their expected molecular weights. Additionally, the average discrepancies between the calculated molecular weights and their nominal values for glycosylated proteins are similar to those for nonglycosylated ones.

Asunto(s)

Bases de Datos de Proteínas , Simulación de Dinámica Molecular , Proteínas/química , Dispersión del Ángulo Pequeño , Programas Informáticos , Difracción de Rayos X , Peso Molecular

RESUMEN

The risk of severe COVID-19 increases with age as older patients are at highest risk. Thus, there is an urgent need to identify how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interacts with blood components during aging. We investigated the whole blood transcriptome from the Genotype-Tissue Expression (GTEx) database to explore differentially expressed genes (DEGs) translated into proteins interacting with viral proteins during aging. From 22 DEGs in aged blood, FASLG, CTSW, CTSE, VCAM1, and BAG3 were associated with immune response, inflammation, cell component and adhesion, and platelet activation/aggregation. Males and females older than 50 years old overexpress FASLG, possibly inducing a hyperinflammatory cascade. The expression of cathepsins (CTSW and CTSE) and the anti-apoptotic co-chaperone molecule BAG3 also increased throughout aging in both genders. By exploring single-cell RNA-sequencing data from peripheral blood of SARS-CoV-2-infected patients, we found FASLG and CTSW expressed in natural killer cells and CD8 + T lymphocytes, whereas BAG3 was expressed mainly in CD4 + T cells, naive T cells, and CD14 + monocytes. In addition, T cell exhaustion was associated with increased expression of CCL4L2 and DUSP4 over blood aging. LAG3, PDCD1, TIGIT, VCAM1, HLA-DRA, and TOX also increased in individuals aged 60-69 years old; conversely, the RGS2 gene decreased with aging. We further identified a distinct gene expression profile associated with type I interferon signaling following blood aging. These results revealed changes in blood molecules potentially related to SARS-CoV-2 infection throughout aging, emphasizing them as therapeutic candidates for aggressive clinical manifestation of COVID-19. KEY MESSAGES: • Prediction of host-viral interactions in the whole blood transcriptome during aging. • Expression levels of FASLG, CTSW, CTSE, VCAM1, and BAG3 increase in aged blood. • Blood interactome reveals targets involved with immune response, inflammation, and blood clots. • SARS-CoV-2-infected patients with high viral load showed FASLG overexpression. • Gene expression profile associated with T cell exhaustion and type I interferon signaling were affected with blood aging.

Asunto(s)

Envejecimiento/sangre , Proteínas Sanguíneas/análisis , COVID-19/genética , SARS-CoV-2/patogenicidad , Transcriptoma , Adulto , Anciano , Envejecimiento/genética , Sangre/metabolismo , Análisis Químico de la Sangre , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/virología , COVID-19/sangre , COVID-19/inmunología , COVID-19/fisiopatología , Fenómenos Fisiológicos Cardiovasculares/genética , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/virología , Estudios de Cohortes , Femenino , Estudios de Asociación Genética , Humanos , Inmunidad Innata/genética , Masculino , Persona de Mediana Edad , Adulto Joven

RESUMEN

The serine/arginine-rich protein kinase 2 (SRPK2) has been reported as upregulated in several cancer types, with roles in hallmarks such as cell migration, growth, and apoptosis. These findings have indicated that SRPK2 is a promising emerging target in drug discovery initiatives. Although high-resolution models are available for SRPK2 (PDB 2X7G), they have been obtained with a heavily truncated recombinant protein version (~50% of the primary structure), due to the presence of long intrinsically unstructured regions. In the present work, we sought to characterize the structure of a full-length recombinant version of SRPK2 in solution. Low-resolution Small-Angle X-ray Scattering data were obtained for both versions of SRPK2. The truncated ΔNΔS-SRPK2 presented a propensity to dimerize at higher concentrations whereas the full-length SRPK2 was mainly found as dimers. The hydrodynamic behavior of the full-length SRPK2 was further investigated by analytical size exclusion chromatography and sedimentation velocity analytical ultracentrifugation experiments. SRPK2 behaved as a monomer-dimer equilibrium and both forms have an elongated shape in solution, pointing to a stretched-to-closed tendency among the conformational plasticity observed. Taken together, these findings allowed us to define unique structural features of the SRPK2 within SRPK family, characterized by its flexible regions outside the bipartite kinase domain.

Asunto(s)

Hidrodinámica , Modelos Moleculares , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes , Conformación Proteica , Proteínas Serina-Treonina Quinasas/genética , Soluciones , Análisis Espectral , Relación Estructura-Actividad

RESUMEN

Enzymes are essential in many biological processes, including second-generation ethanol production. However, enzymes are one of the main expenses for the industrial process in these days. Several studies have been done to maximize cost savings, however, many processes are still economically infeasible. In this study, we report the synthesis of a suspension of lignocresol for recycling or reuse of enzymes in bioprocesses. In this way, it was performed the adsorption assays between lignocresol and ß-glucosidases from Thermotoga petrophila, belonging to the families GH1 and GH3, for the development of a lignocresol-enzyme complex. Our results show that lignocresol maintains greater adsorptive capacity for ß-glucosidases than lignin. This capacity can be explained both by its great hydrophobicity and also by electrostatic characteristics. Therefore, all these results demonstrate good adsorption of the enzymes to the lignocresol, demonstrating great potential for enzymatic recycling.

RESUMEN

Ferulic acid (FA), a low-molecular weight aromatic compound derived from lignin, represents a high-value molecule, used for applications in the cosmetic and pharmaceutical industries. FA can be further enzymatically converted in other commercially interesting molecules, such as vanillin and bioplastics. In several organisms, these transformations often start with a common step of FA activation via CoA-thioesterification, catalyzed by feruloyl-CoA synthetases (Fcs). In this context, these enzymes are of biotechnological interest for conversion of lignin-derived FA into high value chemicals. In this study, we describe the first structural characterization of a prokaryotic Fcs, named FCS1, isolated from a lignin-degrading microbial consortium. The FCS1 optimum pH and temperature were 9 and 37°C, respectively, with Km of 0.12 mM and Vmax of 36.82 U/mg. The circular dichroism spectra indicated a notable secondary structure stability at alkaline pH values and high temperatures. This secondary structure stability corroborates the activity data, which remains high until pH 9. The Small Angle X-Ray Scattering analyses resulted on the tertiary/quaternary structure and the low-resolution envelope in solution of FCS1, which was modeled as a homodimer using the hyperthermophilic nucleoside diphosphate-forming acetyl-CoA synthetase from Candidatus Korachaeum cryptofilum. This study contributes to the field of research by establishing the first biophysical and structural characterization for Fcs, and our data may be used for comparison against novel enzymes of this class that to be studied in the future.

Asunto(s)

Archaea , Proteínas Arqueales , Coenzima A Ligasas , Lignina/química , Metagenoma , Microbiología del Suelo , Archaea/enzimología , Archaea/genética , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Benzaldehídos/química , Benzaldehídos/metabolismo , Coenzima A Ligasas/química , Coenzima A Ligasas/genética , Coenzima A Ligasas/metabolismo , Ácidos Cumáricos/química , Ácidos Cumáricos/metabolismo , Concentración de Iones de Hidrógeno , Lignina/metabolismo , Dominios Proteicos , Suelo

RESUMEN

Lignocellulose feedstock constitutes the most abundant carbon source in the biosphere; however, its recalcitrance remains a challenge for microbial conversion into biofuel and bioproducts. Bacillus licheniformis is a microbial mesophilic bacterium capable of secreting a large number of glycoside hydrolase (GH) enzymes, including a glycoside hydrolase from GH family 9 (BlCel9). Here, we conducted biochemical and biophysical studies of recombinant BlCel9, and its low-resolution molecular shape was retrieved from small angle X-ray scattering (SAXS) data. BlCel9 is an endoglucanase exhibiting maximum catalytic efficiency at pH 7.0 and 60 °C. Furthermore, it retains 80% of catalytic activity within a broad range of pH values (5.5-8.5) and temperatures (up to 50 °C) for extended periods of time (over 48 h). It exhibits the highest hydrolytic activity against phosphoric acid swollen cellulose (PASC), followed by bacterial cellulose (BC), filter paper (FP), and to a lesser extent carboxymethylcellulose (CMC). The HPAEC-PAD analysis of the hydrolytic products demonstrated that the end product of the enzymatic hydrolysis is primarily cellobiose, and also small amounts of glucose, cellotriose, and cellotetraose are produced. SAXS data analysis revealed that the enzyme adopts a monomeric state in solution and has a molecular mass of 65.8 kDa as estimated from SAXS data. The BlCel9 has an elongated shape composed of an N-terminal family 3 carbohydrate-binding module (CBM3c) and a C-terminal GH9 catalytic domain joined together by 20 amino acid residue long linker peptides. The domains are closely juxtaposed in an extended conformation and form a relatively rigid structure in solution, indicating that the interactions between the CBM3c and GH9 catalytic domains might play a key role in cooperative cellulose biomass recognition and hydrolysis.

Asunto(s)

Bacillus licheniformis/enzimología , Bacillus licheniformis/metabolismo , Celulasa/metabolismo , Glicósido Hidrolasas/metabolismo , Lignina/metabolismo , Catálisis , Celobiosa/biosíntesis , Celulosa/análogos & derivados , Celulosa/biosíntesis , Glucosa/biosíntesis , Concentración de Iones de Hidrógeno , Dispersión del Ángulo Pequeño , Tetrosas/biosíntesis , Triosas/biosíntesis , Difracción de Rayos X

RESUMEN

Local myonecrosis is the main event resulting from snakebite envenomation by the Bothrops genus and, frequently, it is not efficiently neutralized by antivenom administration. Proteases, phospholipases A2 (PLA2) and PLA2-like toxins are found in venom related to muscle damage. Functional sites responsible for PLA2-like toxins activity have been proposed recently; they consist of a membrane docking-site and a membrane rupture-site. Herein, a combination of functional, biophysical and crystallographic techniques was used to characterize the interaction between suramin and MjTX-I (a PLA2-like toxin from Bothrops moojeni venom). Functional in vitro neuromuscular assays were performed to study the biological effects of the protein-ligand interaction, demonstrating that suramin neutralizes the myotoxic effect of MjTX-I. Calorimetric assays showed two different binding events: (i) inhibitor-protein interactions and (ii) toxin oligomerization processes. These hypotheses were also corroborated with dynamic light and small angle X-ray scattering assays. The crystal structure of the MjTX-I/suramin showed a totally different interaction mode compared to other PLA2-like/suramin complexes. Thus, we suggested a novel myotoxic mechanism for MjTX-I that may be inhibited by suramin. These results can further contribute to the search for inhibitors that will efficiently counteract local myonecrosis in order to be used as an adjuvant of conventional serum therapy.

Asunto(s)

Fosfolipasas A2/metabolismo , Proteínas de Reptiles/metabolismo , Suramina/química , Animales , Sitios de Unión , Bothrops , Venenos de Crotálidos/metabolismo , Cristalografía por Rayos X , Simulación de Dinámica Molecular , Fosfolipasas A2/química , Estructura Cuaternaria de Proteína , Proteínas de Reptiles/química , Dispersión del Ángulo Pequeño , Suramina/metabolismo , Termodinámica

RESUMEN

Endoglucanases are the main cellulolytic enzymes secreted by the bacterium Xanthomonas campestris pv. campestris (Xcc). The major endoglucanase exported by this bacterium into an external milieu is an enzyme XccCel5A, which belongs to GH5 family subfamily 1 and is encoded by the gene engXCA. We purified XccCel5A using ammonium sulfate precipitation followed by size exclusion chromatography and identified it by zymogram analysis. Circular dichroism and fluorescence spectroscopy studies showed that XccCel5A is stable in a wide pH range and up to about 55°C and denatures at the higher temperatures. The optimal conditions for enzyme activity were identified as T=45°C and pH=7.0. Under the optimum conditions the catalytic efficiency (kcat/KM) of the enzyme was determined as 5.16×10(4)s(-1)M(-1) using carboxymethylcellulose (CMC) as a substrate. Our SAXS studies revealed extended tadpole-shape molecular assembly, typical for cellulases, and allowed to determine an overall shape of the enzyme and a relative position of the catalytic and cellulose binding domains.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Celulasa/química , Celulasa/metabolismo , Xanthomonas campestris/enzimología , Proteínas Bacterianas/genética , Fenómenos Biofísicos , Dominio Catalítico , Celulasa/genética , Dicroismo Circular , Estabilidad de Enzimas , Genes Bacterianos , Cinética , Modelos Moleculares , Conformación Proteica , Dispersión del Ángulo Pequeño , Espectrometría de Fluorescencia , Difracción de Rayos X , Xanthomonas campestris/genética

RESUMEN

Non-productive adsorption of cellulases onto lignins is an important mechanism that negatively affects the enzymatic hydrolysis of lignocellulose biomass. Here, we examined the non-productive adsorption of two bacterial ß-glucosidases (GH1 and GH3) on lignins. The results showed that ß-glucosidases can adsorb to lignins through different mechanisms. GH1 ß-glucosidase adsorption onto lignins was found to be strongly pH-dependent, suggesting that the adsorption is electrostatically modulated. For GH3 ß-glucosidase, the results suggested that the fibronectin type III-like domain interacts with lignins through electrostatic and hydrophobic interactions that can partially, or completely, overcome repulsive electrostatic forces between the catalytic domain and lignins. Finally, the increase of temperature did not result in the increase of ß-glucosidases adsorption, probably because there is no significant increase in hydrophobic regions in the ß-glucosidases structures. The data provided here can be useful for biotechnological applications, especially in the field of plant structural polysaccharides conversion into bioenergy and bioproducts.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Celulasas/química , Celulasas/metabolismo , Lignina/química , Lignina/metabolismo , Adsorción , Biocombustibles , Biomasa , Biotecnología , Dominio de Fibronectina del Tipo III , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Electricidad Estática , Temperatura

RESUMEN

Samples of Eucalyptus urograndis and Eucalyptus grandis sawdust were autohydrolyzed in aqueous conditions to reach temperatures in the range 110-190°C and reaction times of 0-150min in a minireactor. In each minireactor were used a liquor:wood ratio (10:1 L:kg dry wood), in order to assess the effects of the autohydrolysis severity and the crystalline properties of cellulose. The content of extractives, lignin, holocellulose, cellulose, hemicelluloses and crystallinity index obtained from the solid fraction after autohydrolysis of sawdust were determined. This study demonstrated that the hemicelluloses were extensively removed at 170 and 190°C, whereas cellulose was partly degraded to Eucalyptus urograndis and Eucalyptus grandis sawdust. The lignin content decreased, while the extractives content increased. It was defined that during autohydrolysis, had a slight decreased on crystalline structure of cellulose of Eucalyptus urogandis and Eucalyptus grandis.

Asunto(s)

Celulosa/química , Eucalyptus/química , Calor , Reactores Biológicos , Brasil , Hidrólisis , Lignina/química , Agua/análisis , Madera/química

RESUMEN

OBJECTIVES: To biochemically characterize an expansin-like X protein domain from Xanthomonas campestris (XcEXLX1) and to study its synergy with cellulases in cellulose depolymerization. RESULTS: The protein was purified using a combination of ion exchange and size exclusion chromatography rendering about 30 mg pure protein/l culture medium. Circular dichroism spectroscopy and small-angle X-ray scattering studies of XcEXLX1 reveal that it is a strongly disordered ß-sheet protein. Its low resolution envelope fits nicely the crystallographic structure of the homologous protein EXLX1 from Bacillus subtillis. Furthermore, we demonstrate that XcEXLX1 shows a synergistic, pH-dependent effect when combined with a commercial enzymatic preparation (Accellerase 1500), enhancing its hydrolytic activity on a cellulosic substrate. The strongest effect was observed in acid pHs with an increase in sugar release of up to 36 %. CONCLUSION: The synergistic effect arising from the action of the expansin-like protein was considerable in the presence of significantly larger amounts of the commercial enzymatic cocktail then previously observed (0.35 FPU of Accellerase 1500/g substrate).

Asunto(s)

Celulosa/metabolismo , Hidrolasas/aislamiento & purificación , Hidrolasas/metabolismo , Xanthomonas campestris/enzimología , Cromatografía Liquida , Dicroismo Circular , Citosol/química , Concentración de Iones de Hidrógeno , Hidrolasas/química , Hidrólisis , Conformación Proteica , Dispersión del Ángulo Pequeño

RESUMEN

The ß-glucosidases are enzymes essential for several industrial applications, especially in the field of plant structural polysaccharides conversion into bioenergy and bioproducts. In a recent study, we have provided a biochemical characterization of two hyperthermostable ß-glucosidases from Thermotoga petrophila belonging to the families GH1 (TpBGL1) and GH3 (TpBGL3). Here, as part of a continuing investigation, the oligomeric state, the net charge, and the structural stability, at acidic pH, of the TpBGL1 and TpBGL3 were characterized and compared. Enzymatic activity is directly related to the balance between protonation and conformational changes. Interestingly, our results indicated that there were no significant changes in the secondary, tertiary and quaternary structures of the ß-glucosidases at temperatures below 80 °C. Furthermore, the results indicated that both the enzymes are stable homodimers in solution. Therefore, the observed changes in the enzymatic activities are due to variations in pH that modify protonation of the enzymes residues and the net charge, directly affecting the interactions with ligands. Finally, the results showed that the two ß-glucosidases displayed different pH dependence of thermostability at temperatures above 80 °C. TpBGL1 showed higher stability at pH 6 than at pH 4, while TpBGL3 showed similar stability at both pH values. This study provides a useful comparison of the structural stability, at acidic pH, of two different hyperthermostable ß-glucosidases and how it correlates with the activity of the enzymes. The information described here can be useful for biotechnological applications in the biofuel and food industries.

Asunto(s)

Proteínas Bacterianas/química , Celulasas/química , Bacilos Gramnegativos Anaerobios Rectos, Curvos y Espirales/química , Protones , Estabilidad de Enzimas , Bacilos Gramnegativos Anaerobios Rectos, Curvos y Espirales/enzimología , Calor , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Electricidad Estática , Temperatura

RESUMEN

Endo-ß-1, 4-mannanase from Thermotoga petrophila (TpMan) is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5) was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105). In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features.

Asunto(s)

Hidrolasas/química , Modelos Moleculares , Conformación Proteica , Termodinámica , Activación Enzimática , Estabilidad de Enzimas , Glicósidos/metabolismo , Concentración de Iones de Hidrógeno , Hidrolasas/metabolismo , Relación Estructura-Actividad , Temperatura

RESUMEN

Endo-ß-1,4-mannanase from Thermotoga petrophila (TpMan) is a hyperthermostable enzyme that catalyzes the hydrolysis of ß-1,4-mannoside linkages in various mannan-containing polysaccharides. A recent study reported that TpMan is composed of a GH5 catalytic domain joined by a linker to a carbohydrate-binding domain. However, at this moment, there is no three-dimensional structure determined for TpMan. Little is known about the conformation of the TpMan as well as the role of the length and flexibility of the linker on the spatial arrangement of the constitutive domains. In this study, we report the first structural characterization of the entire TpMan by small-angle X-ray scattering combined with the three-dimensional structures of the individual domains in order to shed light on the low-resolution model, overall dimensions, and flexibility of this modular enzyme at different temperatures. The results are consistent with a linker with a compact structure and that occupies a small volume with respect to its large number of amino acids. Furthermore, at 20°C the results are consistent with a model where TpMan is a molecule composed of three distinct domains and that presents some level of molecular flexibility in solution. Even though the full enzyme has some degree of molecular flexibility, there might be a preferable conformation, which could be described by the rigid-body modeling procedure. Finally, the results indicate that TpMan undergoes a temperature-driven transition between conformational states without a significant disruption of its secondary structure. Our results suggest that the linker can optimize the geometry between the other two domains with respect to the substrate at high temperatures. These studies should provide a useful basis for future biophysical studies of entire TpMan.

Asunto(s)

Bacterias/enzimología , Manosidasas/química , Manosidasas/metabolismo , Temperatura , Dicroismo Circular , Dispersión Dinámica de Luz , Concentración de Iones de Hidrógeno , Modelos Moleculares , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Difracción de Rayos X

RESUMEN

Endoglucanases are key enzymes applied to the conversion of biomass aiming for second generation biofuel production. In the present study we obtained the small angle X-ray scattering (SAXS) structure of the G. trabeumendo-1,4-ß-glucanase Cel12A and investigated the influence of an important parameter, temperature, on both secondary and tertiary structure of the enzyme and its activity. The CD analysis for GtCel12A revealed that changes in the CD spectra starts at 55 °C and the Tm calculated from the experimental CD sigmoid curve using the Boltzmann function was 60.2 ± 0.6 °C. SAXS data showed that GtCel12A forms monomers in solution and has an elongated form with a maximum diameter of 60 ± 5 Å and a gyration radius of 19.4 ± 0.1 Å as calculated from the distance distribution function. Kratky analysis revealed that 60 °C is the critical temperature above which we observed clear indications of denaturation. Our results showed the influence of temperature on the stability and activity of enzymes and revealed novel structural features of GtCel12A.

RESUMEN

Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals. In this context, the present study reports the isolation, recombinant expression, biochemical and structural characterization of a novel endoxylanase family GH10 (SCXyl) identified from sugarcane soil metagenome. The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH 6,0 and 45°C. The crystal structure was solved at 2.75 Å resolution, revealing the classical (ß/α)8-barrel fold with a conserved active-site pocket and an inherent flexibility of the Trp281-Arg291 loop that can adopt distinct conformational states depending on substrate binding. The capillary electrophoresis analysis of degradation products evidenced that the enzyme displays unusual capacity to degrade small xylooligosaccharides, such as xylotriose, which is consistent to the hydrophobic contacts at the +1 subsite and low-binding energies of subsites that are distant from the site of hydrolysis. The main reaction products from xylan polymers and phosphoric acid-pretreated sugarcane bagasse (PASB) were xylooligosaccharides, but, after a longer incubation time, xylobiose and xylose were also formed. Moreover, the use of SCXyl as pre-treatment step of PASB, prior to the addition of commercial cellulolytic cocktail, significantly enhanced the saccharification process. All these characteristics demonstrate the advantageous application of this enzyme in several biotechnological processes in food and feed industry and also in the enzymatic pretreatment of biomass for feedstock and ethanol production.

Asunto(s)

Metagenoma/genética , Saccharum/genética , Biotecnología/métodos , Electroforesis Capilar , Endo-1,4-beta Xilanasas/metabolismo , Glucuronatos/metabolismo , Oligosacáridos/metabolismo

RESUMEN

Multifunctional enzyme engineering can improve enzyme cocktails for emerging biofuel technology. Molecular dynamics through structure-based models (SB) is an effective tool for assessing the tridimensional arrangement of chimeric enzymes as well as for inferring the functional practicability before experimental validation. This study describes the computational design of a bifunctional xylanase-lichenase chimera (XylLich) using the xynA and bglS genes from Bacillus subtilis. In silico analysis of the average solvent accessible surface area (SAS) and the root mean square fluctuation (RMSF) predicted a fully functional chimera, with minor fluctuations and variations along the polypeptide chains. Afterwards, the chimeric enzyme was built by fusing the xynA and bglS genes. XylLich was evaluated through small-angle X-ray scattering (SAXS) experiments, resulting in scattering curves with a very accurate fit to the theoretical protein model. The chimera preserved the biochemical characteristics of the parental enzymes, with the exception of a slight variation in the temperature of operation and the catalytic efficiency (kcat/Km). The absence of substantial shifts in the catalytic mode of operation was also verified. Furthermore, the production of chimeric enzymes could be more profitable than producing a single enzyme separately, based on comparing the recombinant protein production yield and the hydrolytic activity achieved for XylLich with that of the parental enzymes.

Asunto(s)

Bacillus subtilis/enzimología , Endo-1,4-beta Xilanasas/química , Glicósido Hidrolasas/química , Simulación de Dinámica Molecular , Proteínas Recombinantes de Fusión/química , Simulación por Computador , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Modelos Moleculares , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Dispersión del Ángulo Pequeño