High-resolution structure of a modular hyperthermostable endo-ß-1,4-mannanase from Thermotoga petrophila: The ancillary immunoglobulin-like module is a thermostabilizing domain.

The endo-ß-1,4-mannanase from the hyperthermostable bacterium Thermotoga petrophila (TpMan) is an enzyme that catalyzes the hydrolysis of mannan and heteromannan polysaccharides. Of the three domains that comprise TpMan, the N-terminal GH5 catalytic domain and the C-terminal carbohydrate-binding domain are connected through a central ancillary domain of unknown structure and function. In this study, we report the partial crystal structure of the TpMan at 1.45 Å resolution, so far, the first modular hyperthermostable endo-ß-1,4-mannanase structure determined. The structure exhibits two domains, a (ß/α)8-barrel GH5 catalytic domain connected via a linker to the central domain with an immunoglobulin-like ß-sandwich fold formed of seven ß-strands. Functional analysis showed that whereas the immunoglobulin-like domain does not have the carbohydrate-binding function, it stacks on the GH5 catalytic domain acting as a thermostabilizing domain and allowing operation at hyperthermophilic conditions. The carbohydrate-binding domain is absent in the crystal structure most likely due to its high flexibility around the immunoglobulin-like domain which may act also as a pivot. These results represent new structural and functional information useful on biotechnological applications for biofuel and food industries.

Systematic studies of the interactions between a model polyphenol compound and microbial ß-glucosidases.

Lignin is a major obstacle for cost-effective conversion of cellulose into fermentable sugars. Non-productive adsorption onto insoluble lignin fragments and interactions with soluble phenols are important inhibition mechanisms of cellulases, including ß-glucosidases. Here, we examined the inhibitory effect of tannic acid (TAN), a model polyphenolic compound, on ß-glucosidases from the bacterium Thermotoga petrophila (TpBGL1 and TpBGL3) and archaeon Pyrococcus furiosus (PfBGL1). The results revealed that the inhibition effects on ß-glucosidases were TAN concentration-dependent. TpBGL1 and TpBGL3 were more tolerant to the presence of TAN when compared with PfBGL1, while TpBGL1 was less inhibited when compared with TpBGL3. In an attempt to better understand the inhibitory effect, the interaction between TAN and ß-glucosidases were analyzed by isothermal titration calorimetry (ITC). Furthermore, the exposed hydrophobic surface areas in ß-glucosidases were analyzed using a fluorescent probe and compared with the results of inhibition and ITC. The binding constants determined by ITC for the interactions between TAN and ß-glucosidases presented the same order of magnitude. However, the number of binding sites and exposed hydrophobic surface areas varied for the ß-glucosidases studied. The binding between TAN and ß-glucosidases were driven by enthalpic effects and with an unfavorable negative change in entropy upon binding. Furthermore, the data suggest that there is a high correlation between exposed hydrophobic surface areas and the number of binding sites on the inhibition of microbial ß-glucosidases by TAN. These studies can be useful for biotechnological applications.

Photobiosynthesis of stable and functional silver/silver chloride nanoparticles with hydrolytic activity using hyperthermophilic ß-glucosidases with industrial potential.

The ß-glucosidases are important enzymes employed in a large number of processes and industrial applications, including biofuel production from biomass. Therefore, in this study, we reported for the first time the photobiosynthesis of stable and functional silver/silver chloride nanoparticles (Ag/AgCl-NPs) using two hyperthermostable bacterial ß-glucosidases with industrial potential. The syntheses were straightforward and rapid processes carried out by mixing ß-glucosidase and silver nitrate (in buffer 10mM Tris-HCl, pH 8) under irradiation with light (over a wavelength range of 450-600nm), therefore, compatible with the green chemistry procedure. Synthesized Ag/AgCl-NPs were characterized using a series of physical techniques. Absorption spectroscopy showed a strong absorption band centered at 460nm due to surface plasmon resonance of the Ag-NPs. X-ray diffraction analysis revealed that the Ag/AgCl-NPs were purely crystalline in nature. Under electron microscopy, Ag/AgCl-NPs of variable diameter ranging from 10 to 100nm can be visualized. Furthermore, electron microscopy, zeta potential and Fourier transform infrared spectroscopy results confirmed the presence of ß-glucosidases coating and stabilizing the Ag/AgCl-NPs. Finally, the results showed that the enzymatic activities were maintained in the ß-glucosidases assisted Ag/AgCl-NPs. The information described here should provide a useful basis for future studies of ß-glucosidases assisted Ag/AgCl-NPs, including biotechnological applications.

Non-productive adsorption of bacterial ß-glucosidases on lignins is electrostatically modulated and depends on the presence of fibronection type III-like domain.

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.

Chemical stability of a cold-active cellulase with high tolerance toward surfactants and chaotropic agent.

CelE1 is a cold-active endo-acting glucanase with high activity at a broad temperature range and under alkaline conditions. Here, we examined the effects of pH on the secondary and tertiary structures, net charge, and activity of CelE1. Although variation in pH showed a small effect in the enzyme structure, the activity was highly influenced at acidic conditions, while reached the optimum activity at pH 8. Furthermore, to estimate whether CelE1 could be used as detergent additives, CelE1 activity was evaluated in the presence of surfactants. Ionic and nonionic surfactants were not able to reduce CelE1 activity significantly. Therefore, CelE1 was found to be promising candidate for use as detergent additives. Finally, we reported a thermodynamic analysis based on the structural stability and the chemical unfolding/refolding process of CelE1. The results indicated that the chemical unfolding proceeds as a reversible two-state process. These data can be useful for biotechnological applications.

Oligomeric state and structural stability of two hyperthermophilic ß-glucosidases from Thermotoga petrophila.

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.

Conformational changes in a hyperthermostable glycoside hydrolase: enzymatic activity is a consequence of the loop dynamics and protonation balance.

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.

Modular hyperthermostable bacterial endo-ß-1,4-mannanase: molecular shape, flexibility and temperature-dependent conformational changes.

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.