RESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Bacilos Gramnegativos Anaerobios Rectos, Curvos y Espirales/enzimología , Pyrococcus furiosus/enzimología , Taninos/farmacología , beta-Glucosidasa/metabolismo , Proteínas Arqueales/antagonistas & inhibidores , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Calorimetría , Relación Dosis-Respuesta a Droga , Escherichia coli , Bacilos Gramnegativos Anaerobios Rectos, Curvos y Espirales/química , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Unión Proteica , Pyrococcus furiosus/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tensoactivos/farmacología , beta-Glucosidasa/antagonistas & inhibidores , beta-Glucosidasa/química , beta-Glucosidasa/genéticaRESUMEN
A solution molecular model for the conformationally dynamically heterogeneous Pyrococcus furiosus ferredoxin with an intact disulfide bond has been constructed on the basis of reported (1)H NMR spectral parameters using distance geometry and simulated annealing protocols. Conventional long-mixing time NOESY and H-bonding constraints have been augmented by previously reported short-mixing time NOESY, steady-state NOE, and cluster paramagnetism-induced relaxation. The family of 15 structures with inconsequential violations exhibited low rms deviations for backbone atoms for the overwhelming majority of the residues, including the cluster ligating loop with the unprecedented ligated Asp14. Larger rms deviations were observed across the disulfide bond, but closer inspection revealed that the 15 structures can be factored into 10 substructures exhibiting an "S" or right-handed disulfide orientation and 5 exhibiting an "R" or left-handed disulfide orientation. The remainder of the structure is indistinguishable for the two disulfide orientations but confirms stabilizing extensions of secondary structural elements in the lengthening of the long helix and both the lengthening and incorporation of a third strand into the beta-sheet involving the termini, with these extensions interacting strongly in a modular fashion through the rings of Tyr46 and Trp2. These extensions of stabilizing interactions in Pyrococcus furiosus Fd, however, lead to strong destabilization of the disulfide bond and destabilization of the highly conserved first and last beta-turns in the sequence. It is concluded that the structural alternations in Pyrococcus Fd relative to other hyperthermostable Fds are not to increase thermostability but to place "stress" on the disulfide bond and render it more reducible. The possible physiological implications of this unique reducible disulfide bond are discussed.