RESUMEN
Enzyme reactions, both in Nature and technical applications, commonly occur at the interface of immiscible phases. Nevertheless, stringent descriptions of interfacial enzyme catalysis remain sparse, and this is partly due to a shortage of coherent experimental data to guide and assess such work. In this work, we produced and kinetically characterized 83 cellulases, which revealed a conspicuous linear free energy relationship (LFER) between the substrate binding strength and the activation barrier. The scaling occurred despite the investigated enzymes being structurally and mechanistically diverse. We suggest that the scaling reflects basic physical restrictions of the hydrolytic process and that evolutionary selection has condensed cellulase phenotypes near the line. One consequence of the LFER is that the activity of a cellulase can be estimated from its substrate binding strength, irrespectively of structural and mechanistic details, and this appears promising for in silico selection and design within this industrially important group of enzymes.
Asunto(s)
Algoritmos , Celulasas/metabolismo , Celulosa/metabolismo , Simulación de Dinámica Molecular , Biocatálisis , Celulasas/química , Hidrólisis , Cinética , Unión Proteica , Dominios Proteicos , Especificidad por SustratoRESUMEN
Lytic polysaccharide monooxygenases (LPMOs) are known to act synergistically with glycoside hydrolases in industrial cellulolytic cocktails. However, a few studies have reported severe impeding effects of C1-oxidizing LPMOs on the activity of reducing-end cellobiohydrolases. The mechanism for this effect remains unknown, but it may have important implications as reducing-end cellobiohydrolases make up a significant part of such cocktails. To elucidate whether the impeding effect is general for different reducing-end cellobiohydrolases and study the underlying mechanism, we conducted a comparative biochemical investigation of the cooperation between a C1-oxidizing LPMO from Thielavia terrestris and three reducing-end cellobiohydrolases; Trichoderma reesei (TrCel7A), T. terrestris (TtCel7A), and Myceliophthora heterothallica (MhCel7A). The enzymes were heterologously expressed in the same organism and thoroughly characterized biochemically. The data showed distinct differences in synergistic effects between the LPMO and the cellobiohydrolases; TrCel7A was severely impeded, TtCel7A was moderately impeded, while MhCel7A was slightly boosted by the LPMO. We investigated effects of C1-oxidations on cellulose chains on the activity of the cellobiohydrolases and found reduced activity against oxidized cellulose in steady-state and pre-steady-state experiments. The oxidations led to reduced maximal velocity of the cellobiohydrolases and reduced rates of substrate complexation. The extent of these effects differed for the cellobiohydrolases and scaled with the extent of the impeding effect observed in the synergy experiments. Based on these results, we suggest that C1-oxidized chain ends are poor attack sites for reducing-end cellobiohydrolases. The severity of the impeding effects varied considerably among the cellobiohydrolases, which may be relevant to consider for optimization of industrial cocktails.
Asunto(s)
Celulosa 1,4-beta-Celobiosidasa/metabolismo , Celulosa/metabolismo , Proteínas Fúngicas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Polisacáridos/metabolismo , Hidrólisis , Hypocreales/enzimología , Oxidación-Reducción , Polisacáridos/química , Sordariales/enzimologíaRESUMEN
Cellobiohydrolases effectively degrade cellulose and are of biotechnological interest because they can convert lignocellulosic biomass to fermentable sugars. Here, we implemented a fluorescence-based method for real-time measurements of complexation and decomplexation of the processive cellulase Cel7A and its insoluble substrate, cellulose. The method enabled detailed kinetic and thermodynamic analyses of ligand binding in a heterogeneous system. We studied WT Cel7A and several variants in which one or two of four highly conserved Trp residues in the binding tunnel had been replaced with Ala. WT Cel7A had on/off-rate constants of 1 × 105 m-1 s-1 and 5 × 10-3 s-1, respectively, reflecting the slow dynamics of a solid, polymeric ligand. Especially the off-rate constant was many orders of magnitude lower than typical values for small, soluble ligands. Binding rate and strength both were typically lower for the Trp variants, but effects of the substitutions were moderate and sometimes negligible. Hence, we propose that lowering the activation barrier for complexation is not a major driving force for the high conservation of the Trp residues. Using so-called Φ-factor analysis, we analyzed the kinetic and thermodynamic results for the variants. The results of this analysis suggested a transition state for complexation and decomplexation in which the reducing end of the ligand is close to the tunnel entrance (near Trp-40), whereas the rest of the binding tunnel is empty. We propose that this structure defines the highest free-energy barrier of the overall catalytic cycle and hence governs the turnover rate of this industrially important enzyme.
Asunto(s)
Celulasa/metabolismo , Celulosa/metabolismo , Proteínas Fúngicas/metabolismo , Trichoderma/metabolismo , Triptófano/metabolismo , Dominio Catalítico , Celulasa/química , Activación Enzimática , Proteínas Fúngicas/química , Cinética , Modelos Moleculares , Unión Proteica , Especificidad por Sustrato , Termodinámica , Trichoderma/química , Triptófano/químicaRESUMEN
Understanding the pH effect of cellulolytic enzymes is of great technological importance. In this study, we have examined the influence of pH on activity and stability for central cellulases (Cel7A, Cel7B, Cel6A from Trichoderma reesei, and Cel7A from Rasamsonia emersonii). We systematically changed pH from 2 to 7, temperature from 20°C to 70°C, and used both soluble (4-nitrophenyl ß- d-lactopyranoside [pNPL]) and insoluble (Avicel) substrates at different concentrations. Collective interpretation of these data provided new insights. An unusual tolerance to acidic conditions was observed for both investigated Cel7As, but only on real insoluble cellulose. In contrast, pH profiles on pNPL were bell-shaped with a strong loss of activity both above and below the optimal pH for all four enzymes. On a practical level, these observations call for the caution of the common practice of using soluble substrates for the general characterization of pH effects on cellulase activity. Kinetic modeling of the experimental data suggested that the nucleophile of Cel7A experiences a strong downward shift in pKa upon complexation with an insoluble substrate. This shift was less pronounced for Cel7B, Cel6A, and for Cel7A acting on the soluble substrate, and we hypothesize that these differences are related to the accessibility of water to the binding region of the Michaelis complex.
Asunto(s)
Celulasas/química , Celulasas/metabolismo , Celulosa/química , Celulosa/metabolismo , Sitios de Unión , Celulasas/genética , Estabilidad de Enzimas , Eurotiales/enzimología , Eurotiales/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentración de Iones de Hidrógeno , Hypocreales/enzimología , Hypocreales/genética , Cinética , Modelos Moleculares , Unión Proteica , TemperaturaRESUMEN
Glycoside hydrolase family 7 (GH7) cellulases are some of the most efficient degraders of cellulose, making them particularly relevant for industries seeking to produce renewable fuels from lignocellulosic biomass. The secretome of the cellulolytic model fungus Trichoderma reesei contains two GH7s, termed TrCel7A and TrCel7B. Despite having high structural and sequence similarities, the two enzymes are functionally quite different. TrCel7A is an exolytic, processive cellobiohydrolase (CBH), with high activity on crystalline cellulose, whereas TrCel7B is an endoglucanase (EG) with a preference for more amorphous cellulose. At the structural level, these functional differences are usually ascribed to the flexible loops that cover the substrate-binding areas. TrCel7A has an extensive tunnel created by eight peripheral loops, and the absence of four of these loops in TrCel7B makes its catalytic domain a more open cleft. To investigate the structure-function relationships of these loops, here we produced and kinetically characterized several variants in which four loops unique to TrCel7A were individually deleted to resemble the arrangement in the TrCel7B structure. Analysis of a range of kinetic parameters consistently indicated that the B2 loop, covering the substrate-binding subsites -3 and -4 in TrCel7A, was a key determinant for the difference in CBH- or EG-like behavior between TrCel7A and TrCel7B. Conversely, the B3 and B4 loops, located closer to the catalytic site in TrCel7A, were less important for these activities. We surmise that these results could be useful both in further mechanistic investigations and for guiding engineering efforts of this industrially important enzyme family.
Asunto(s)
Secuencia de Aminoácidos , Celulosa 1,4-beta-Celobiosidasa , Proteínas Fúngicas , Eliminación de Secuencia , Trichoderma , Celulosa 1,4-beta-Celobiosidasa/química , Celulosa 1,4-beta-Celobiosidasa/genética , Cristalografía por Rayos X , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Estructura Secundaria de Proteína , Trichoderma/enzimología , Trichoderma/genéticaRESUMEN
We have measured activity and substrate affinity of the thermostable cellobiohydrolase, Cel7A, from Rasamsonia emersonii over a broad range of temperatures. For the wild type enzyme, which does not have a Carbohydrate Binding Module (CBM), higher temperature only led to moderately increased activity against cellulose, and we ascribed this to a pronounced, temperature induced desorption of enzyme from the substrate surface. We also tested a "high affinity" variant of R. emersonii Cel7A with a linker and CBM from a related enzyme. At room temperature, the activity of the variant was similar to the wild type, but the variant was more accelerated by temperature and about two-fold faster around 70 °C. This better thermoactivation of the high-affinity variant could not be linked to differences in stability or the catalytic process, but coincided with less desorption as temperature increased. Based on these observations and earlier reports on moderate thermoactivation of cellulases, we suggest that better cellulolytic activity at industrially relevant temperatures may be attained by engineering improved substrate affinity into enzymes that already possess good thermostability.