RESUMEN
Degrading cellulose is a key step in the processing of lignocellulosic biomass into bioethanol. Cellobiose, the disaccharide product of cellulose degradation, has been shown to inhibit cellulase activity, but the mechanisms underlying product inhibition are not clear. We combined single-molecule imaging and biochemical investigations with the goal of revealing the mechanism by which cellobiose inhibits the activity of Trichoderma reesei Cel7A, a well-characterized exo-cellulase. We find that cellobiose slows the processive velocity of Cel7A and shortens the distance moved per encounter; effects that can be explained by cellobiose binding to the product release site of the enzyme. Cellobiose also strongly inhibits the binding of Cel7A to immobilized cellulose, with a Ki of 2.1 mM. The isolated catalytic domain (CD) of Cel7A was also inhibited to a similar degree by cellobiose, and binding of an isolated carbohydrate-binding module to cellulose was not inhibited by cellobiose, suggesting that cellobiose acts on the CD alone. Finally, cellopentaose inhibited Cel7A binding at micromolar concentrations without affecting the enzyme's velocity of movement along cellulose. Together, these results suggest that cellobiose inhibits Cel7A activity both by binding to the "back door" product release site to slow activity and to the "front door" substrate-binding tunnel to inhibit interaction with cellulose. These findings point to strategies for engineering cellulases to reduce product inhibition and enhance cellulose degradation, supporting the growth of a sustainable bioeconomy.
Asunto(s)
Celobiosa , Celulasa , Celulosa , Hypocreales , Celobiosa/metabolismo , Celulasa/metabolismo , Celulasa/antagonistas & inhibidores , Celulosa/metabolismo , Hypocreales/enzimología , Hypocreales/metabolismo , Imagen Individual de Molécula/métodos , Dominio Catalítico , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/químicaRESUMEN
BACKGROUND: Silicon and aluminium oxides make the bulk of agricultural soils. Plants absorb dissolved silicon as silicic acid into their bodies through their roots. The silicic acid moves with transpiration to target tissues in the plant body, where it polymerizes into biogenic silica. Mostly, the mineral forms on a matrix of cell wall polymers to create a composite material. Historically, silica deposition (silicification) was supposed to occur once water evaporated from the plant surface, leaving behind an increased concentration of silicic acid within plant tissues. However, recent publications indicate that certain cell wall polymers and proteins initiate and control the extent of plant silicification. SCOPE: Here we review recent publications on the polymers that scaffold the formation of biogenic plant silica, and propose a paradigm shift from spontaneous polymerization of silicic acid to dedicated active metabolic processes that control both the location and the extent of the mineralization. CONCLUSION: Protein activity concentrates silicic acid beyond its saturation level. Polymeric structures at the cell wall stabilize the supersaturated silicic acid and allow its flow with the transpiration stream, or bind it and allow its initial condensation. Silica nucleation and further polymerization are enabled on a polymeric scaffold, which is embedded within the mineral. Deposition is terminated once free silicic acid is consumed or the chemical moieties for its binding are saturated.
Asunto(s)
Ácido Silícico , Dióxido de Silicio , Dióxido de Silicio/metabolismo , Ácido Silícico/química , Ácido Silícico/metabolismo , Silicio/metabolismo , Plantas/metabolismo , PolímerosRESUMEN
Hydrated silica (SiO2·nH2O) aggregates in the root endodermis of grasses. Application of soluble silicates (Si) to roots is associated with variations in the balance of reactive oxygen species (ROS), increased tolerance to a broad range of stresses affecting ROS concentrations, and early lignin deposition. In sorghum (Sorghum bicolor L.), silica aggregation is patterned in an active silicification zone (ASZ) by a special type of aromatic material forming a spotted pattern. The deposition has a signature typical of lignin. Since lignin polymerization is mediated by ROS, we studied the formation of root lignin and silica controlled by ROS via modulating hydrogen peroxide (H2O2) concentrations in the growth medium. Sorghum seedlings were grown hydroponically and supplemented with Si, H2O2, and KI, an ionic compound that catalyses H2O2 decomposition. Lignin and silica deposits in the endodermis were studied by histology, scanning electron and Raman microscopies. Cell wall composition was quantified by thermal gravimetric analysis. Endodermal H2O2 concentration correlated to the extent of lignin-like deposition along the root, but did not affect its patterning in spots. Our results show that the ASZ spots were necessary for root silica aggregation, and suggest that silicification is intensified under oxidative stress as a result of increased ASZ lignin-like deposition.
Asunto(s)
Sorghum , Peróxido de Hidrógeno , Raíces de Plantas , Plantones , Dióxido de SilicioRESUMEN
Silicon dioxide in the form of hydrated silica is a component of plant tissues that can constitute several percent by dry weight in certain taxa. Nonetheless, the mechanism of plant silica formation is mostly unknown. Silicon (Si) is taken up from the soil by roots in the form of monosilicic acid molecules. The silicic acid is carried in the xylem and subsequently polymerizes in target sites to silica. In roots of sorghum (Sorghum bicolor), silica aggregates form in an orderly pattern along the inner tangential cell walls of endodermis cells. Using Raman microspectroscopy, autofluorescence, and scanning electron microscopy, we investigated the structure and composition of developing aggregates in roots of sorghum seedlings. Putative silica aggregation loci were identified in roots grown under Si starvation. These micrometer-scale spots were constructed of tightly packed modified lignin, and nucleated trace concentrations of silicic acid. Substantial variation in cell wall autofluorescence between Si+ and Si- roots demonstrated the impact of Si on cell wall chemistry. We propose that in Si- roots, the modified lignin cross-linked into the cell wall and lost its ability to nucleate silica. In Si+ roots, silica polymerized on the modified lignin and altered its structure. Our work demonstrates a high degree of control over lignin and silica deposition in cell walls.
Asunto(s)
Sorghum , Pared Celular , Lignina , Raíces de Plantas , Dióxido de SilicioRESUMEN
Silica aggregates at the endodermis of sorghum roots. Aggregation follows a spotted pattern of locally deposited lignin at the inner tangential cell walls. Autofluorescence microscopy suggests that non-silicified (-Si) lignin spots are composed of two distinct concentric regions of varied composition. To highlight variations in lignin chemistry, we used Raman microspectroscopy to map the endodermal cell wall and silica aggregation sites in sorghum roots grown hydroponically with or without Si amendment. In +Si samples, the aggregate center was characterized by typical lignin monomer bands surrounded by lignin with a low level of polymerization. Farther from the spot, polysaccharide concentration increased and soluble silicic acid was detected in addition to silica bands. In -Si samples, the main band at the spot center was assigned to lignin radicals and highly polymerized lignin. Both +Si and -Si loci were enriched by aromatic carbonyls. We propose that at silica aggregation sites, carbonyl rich lignin monomers are locally exported to the apoplast. These monomers are radicalized and polymerized into short lignin polymers. In the presence of silicic acid, bonds typically involved in lignin extension, bind to silanols and nucleate silica aggregates near the monomer extrusion loci. This process inhibits further polymerization of lignin. In -Si samples, the monomers diffuse farther in the wall and crosslink with cell wall polymers, forming a ring of dense lignified cell wall around their export sites.
RESUMEN
BACKGROUND: Cellulose degradation by cellulases has been studied for decades due to the potential of using lignocellulosic biomass as a sustainable source of bioethanol. In plant cell walls, cellulose is bonded together and strengthened by the polyphenolic polymer, lignin. Because lignin is tightly linked to cellulose and is not digestible by cellulases, is thought to play a dominant role in limiting the efficient enzymatic degradation of plant biomass. Removal of lignin via pretreatments currently limits the cost-efficient production of ethanol from cellulose, motivating the need for a better understanding of how lignin inhibits cellulase-catalyzed degradation of lignocellulose. Work to date using bulk assays has suggested three possible inhibition mechanisms: lignin blocks access of the enzyme to cellulose, lignin impedes progress of the enzyme along cellulose, or lignin binds cellulases directly and acts as a sink. RESULTS: We used single-molecule fluorescence microscopy to investigate the nanoscale dynamics of Cel7A from Trichoderma reesei, as it binds to and moves along purified bacterial cellulose in vitro. Lignified cellulose was generated by polymerizing coniferyl alcohol onto purified bacterial cellulose, and the degree of lignin incorporation into the cellulose meshwork was analyzed by optical and electron microscopy. We found that Cel7A preferentially bound to regions of cellulose where lignin was absent, and that in regions of high lignin density, Cel7A binding was inhibited. With increasing degrees of lignification, there was a decrease in the fraction of Cel7A that moved along cellulose rather than statically binding. Furthermore, with increasing lignification, the velocity of processive Cel7A movement decreased, as did the distance that individual Cel7A molecules moved during processive runs. CONCLUSIONS: In an in vitro system that mimics lignified cellulose in plant cell walls, lignin did not act as a sink to sequester Cel7A and prevent it from interacting with cellulose. Instead, lignin both blocked access of Cel7A to cellulose and impeded the processive movement of Cel7A along cellulose. This work implies that strategies for improving biofuel production efficiency should target weakening interactions between lignin and cellulose surface, and further suggest that nonspecific adsorption of Cel7A to lignin is likely not a dominant mechanism of inhibition.