Biology subjects

Haviland, Z. K.

Publications and source records attributed to Haviland, Z. K..

5 recordsLinked to original sources

bioRxiv2024

Xylan inhibition of cellulase binding and processivity observed at single-molecule resolution

Efficient cellulose degradation by cellulase enzymes is crucial for using lignocellulosic biomass in bioenergy production. In the cell wall of plants, cellulose is bound by lignin and hemicellulose, which are key factors contributing to the recalcitrance of plant biomass. These non-cellulosic cell wall components are known to interfere with the function of cellulolytic enzymes. While the effects of lignin have been studied extensively, the contribution of xylan, the major hemicellulose in the secondary cell walls of plants, is often overlooked. To study those effects, we generated model cell wall composites by growing bacterial cellulose supplemented with varying concentrations of purified xylan. We used single-molecule microscopy to image and track fluorescently labeled TrCel7A, a commonly used model cellulase, as it binds and hydrolyses cellulose in these synthetic composites. We found that minute amounts of xylan are sufficient to significantly inhibit the binding of Cel7A to cellulose. The inclusion of xylan also reduced considerably the proportion of moving enzyme molecules, without affecting their velocity and run length. We suggest that, when available at low concentrations, xylan thinly coats cellulose fibrils, and incorporates as continuous patches when available at higher concentrations. Non-productive binding of Cel7A to xylan was not found to be a major inhibition mechanism. Our results highlight the importance of targeting xylan removal during biomass processing and demonstrate the potential of using single-molecule imagining to study the activity and limitations of cellulolytic enzymes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/577775v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1c7afbeorg.highwire.dtl.DTLVardef@e30ce4org.highwire.dtl.DTLVardef@635358org.highwire.dtl.DTLVardef@f4bdc9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Lignin impairs Cel7A degradation of cellulose by impeding enzyme movement and not by acting as a sink

BackgroundCellulose 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. ResultsWe 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. ConclusionsIn 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.

plant biology↗

bioRxiv2023

Single-molecule tracking reveals dual front door/back door inhibition of Cel7A cellulase by its product cellobiose

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 decreases the binding rate of Cel7A to immobilized cellulose but does not slow the binding rate of an isolated carbohydrate-binding module, suggesting that cellobiose inhibits binding of the catalytic domain of Cel7A to cellulose. In support of this, cellopentaose, which is considerably larger than cellobiose, also slows the binding rate of Cel7A to cellulose without affecting the velocity and run length. 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 new strategies for engineering cellulases to reduce product inhibition and enhance cellulose degradation, supporting the growth of a sustainable bioeconomy. SignificanceCellulose, a polymer of repeating glucose subunits, is the primary component of plant cell walls. A promising route to reducing petrochemical use is digesting plant biomass to glucose and fermenting glucose to bioethanol. Cel7A is a model cellulase enzyme that degrades cellulose from one end to generate the disaccharide product, cellobiose. Because industrial-scale bioethanol generation generates high concentrations of cellobiose, product inhibition is a significant concern. We investigated product inhibition of Cel7A by cellobiose at the single-molecule level and found that cellobiose both slows the movement of Cel7 along cellulose and inhibits the initial binding of Cel7 to cellulose. These results suggest that cellobiose binds to the enzyme at more than one site and achieves its inhibition by multiple mechanisms.

plant biology↗

bioRxiv2021

An integrated multi-wavelength SCATTIRSTORM microscope combining TIRFM and IRM modalities for imaging cellulases and other processive enzymes

We describe a multimodal SCATTIRSTORM microscope for visualizing processive enzymes moving on immobilized substrates. The instrument combines Interference Reflection Microscopy (IRM) with multi-wavelength Total Internal Reflectance Fluorescence Microscopy (TIRFM). The microscope can localize quantum dots with a precision of 2.8 nm at 100 frames/s, and was used to image the dynamics of the cellulase, Cel7a interacting surface-immobilized cellulose. The instrument, which was built with off-the-shelf components and controlled by custom software, is suitable for tracking other degradative enzymes such as collagenases, as well as motor proteins moving along immobilized tracks.

biophysics↗

bioRxiv2021

Nanoscale dynamics of cellulase TrCel7A digesting cellulose

Understanding how cellulases catalyze the digestion of lignocellulose is a major goal of bioenergy research. Cel7A from Trichoderma reesei is a model exoglucanase that degrades cellulose strands from their reducing ends by processively cleaving individual cellobiose units. Despite being one of the most studied cellulases, the binding and hydrolysis mechanisms of Cel7A are still debated. We used single-molecule tracking to analyze the dynamics of 11,116 quantum dot-labeled TrCel7A binding to and moving processively along immobilized Gluconoacetobacter cellulose. Enzyme molecules were localized with a spatial precision of a few nanometers and followed for hundreds of seconds. Most enzymes bound into a static state and dissociated without detectable movement. Processive enzymes moved an average distance of 39 nm at an average speed of 3.2 nm/s. Static binding episodes preceding and following processive runs were of similar duration to static binding events that lacked any processive movement. Transient jumps of >20 nm were observed, but no diffusive behavior indicative of a diffusive search of the enzyme for a free cellulose strand end was observed. These data were integrated into a three-state model in which TrCel7A molecules can bind from solution into either a static or a processive state, and can reversibly switch between static and processive states before dissociating. From these results, we conclude that the rate-limiting step for cellulose degradation by Cel7A is the transition out of the static state either by dissociation from the cellulose surface or initiation of a processive run.

plant biology↗