David Wilson

Cornell University | CU · Department of Molecular Biology and Genetics

Glycoside hydrolase (GH) family 48 is an understudied and increasingly important exoglucanase family found in the majority of bacterial cellulase systems. Moreover, many thermophilic enzyme systems contain GH48 enzymes. Deletion of GH48 enzymes in these microorganisms results in drastic reduction in biomass deconstruction. Surprisingly, given their...

Background Auxiliary activity (AA) enzymes are produced by numerous bacterial and fungal species to assist in the degradation of biomass. These enzymes are abundant but have yet to be fully characterized. Here, we report the X-ray structure of Thermobifida fusca AA10A (TfAA10A), investigate mutational characterization of key surface residues near i...

Even though only a small fraction of bacterial species can grow on cellulose as a sole carbon source, there is extensive diversity among cellulolytic bacteria for aerobic and anaerobic species. Out of 29 cultured bacterial phyla, 6 contain cellulolytic species. About half of the known cellulolytic bacteria are saprophytes that degrade dead plant ma...

Importance: The bacterium Cytophaga hutchinsonii digests crystalline cellulose by an unknown mechanism. It lacks processive cellobiohydrolases that are often involved in cellulose digestion. Critical cellulolytic enzymes were identified by genetic analyses. Intracellular (periplasmic) nonprocessive endoglucanases performed an important role in cel...

Auxiliary activity (AA) enzymes are produced by numerous bacterial and fungal species to assist in degrading biomass substrates. Although abundant, these enzymes have not been fully explored. The recently characterized AA10 family of bacterial lytic polysaccharide monooxygenases (LPMOs) performs oxidative cleavage of crystalline polysaccharide subs...

Cellulases are currently the third largest industrial enzyme class in sales volume and are likely to become the largest when and if the cellulosic ethanol industry matures. Cellulases also are key enzymes in recycling plant cell walls in nature. They are very diverse enzymes, because there are at least 10 different protein folds for cellulases, and...

Molecular dynamics (MD) simulations were used to study substrate recognition by the family 48 exocellulase CelF from Clostridium cellulolyticum. It was hypothesized that residues around the entrance of the active site tunnel of this enzyme might serve to recognize and bind the substrate through an affinity for the cellulose monomer repeat unit, β-D...

Thermobifida fusca Cel6A is an endoglucanase with relatively high activity on crystalline cellulose. Trp 231 is a substrate-binding residue in the −2 sub-site that is within the active-site cleft. Mutating Trp 231 to Ala, Asp, or His causes significant decreases in activity on both oligosaccharides and cellulose. The largest decreases in activity a...

Lignocellulosic biomass is a potential source of sustainable transportation fuels, but efficient enzymatic saccharification of cellulose is a key challenge in its utilization. Cellulases from the glycoside hydrolase (GH) family 48 constitute an important component of bacterial biomass degrading systems and structures of three enzymes from this fami...

Molecular dynamics simulations were used to study the possible catalytic role of an unusual conserved water-filled pore structure in the family 48 cellulase enzyme Cel48A from Thermobifida fusca. It was hypothesized that this pore serves as the pathway for the water molecules consumed in the hydrolysis catalyzed by the enzyme to reach the active si...

Lignocellulosic biomass is a potential source of sustainable transportation fuels, but efficient enzymatic saccharification of cellulose is a key challenge in its utilization. Cellulases from the glycoside hydrolase (GH) family 48 constitute an important component of bacterial biomass degrading systems and structures of three enzymes from this fami...

Lignocellulosic biomass is digested in nature by the synergistic activities of enzymes with complementary properties and understanding synergistic interactions will improve the efficiency of industrial biomass use for sustainable fuels and chemicals. TfCel9A and TfCel48A from a model bacterium Thermobifida fusca are two cellulases with different pr...

Cellobiohydrolases (CBHs) are typically major components of natural enzyme cocktails for biomass degradation. Their active sites are enclosed in a tunnel, enabling processive hydrolysis of cellulose chains. Glycoside hydrolase Family 6 (GH6) CBHs act from non-reducing ends by an inverting mechanism, and are present in many cellulolytic fungi and ba...

Lignocellulosic biomass is a potential source of renewable, low-carbon-footprint liquid fuels. Biomass recalcitrance and enzyme cost are key challenges associated with the large-scale production of cellulosic fuel. Kinetic modeling of enzymatic cellulose digestion has been complicated by the heterogeneous nature of the substrate and by the fact tha...

Lignocellulosic biomass, the most abundant polymer on Earth, is typically composed of three major constituents: cellulose, hemicellulose, and lignin. The crystallinity of cellulose, hydrophobicity of lignin, and encapsulation of cellulose by the lignin-hemicellulose matrix are three major factors that contribute to the observed recalcitrance of lig...

Background Microorganisms employ a multiplicity of enzymes to efficiently degrade the composite structure of plant cell wall cellulosic polysaccharides. These remarkable enzyme systems include glycoside hydrolases (cellulases, hemicellulases), polysaccharide lyases, and the carbohydrate esterases. To accomplish this challenging task, several strate...

There are two types of processive cellulases, exocellulases and processive endoglucanases. There are also two classes of exocellulases, ones that attack the reducing ends of cellulose chains and ones that attack the nonreducing ends. There are a number of ways of assaying processivity but none of them are ideal. It appears that exocellulases, all o...

β-Xylosidases are hemicellulases that hydrolyze short xylo-oligosaccharides into xylose units, thus complementing endoxylanase degradation of the hemicellulose component of lignocellulosic substrates. Here, we describe the cloning, characterization, and kinetic analysis of a glycoside hydrolase family 43 β-xylosidase (Xyl43A) from the aerobic cellu...

Cellulases acting on crystalline cellulose in synergistic mixtures have higher combined activities than the sum of their individual activities. The mechanisms by which different types of cellulases enhance each other's activities are complex and not completely understood, and the published data is often inconsistent. We discuss the role of the subs...

Detailed understanding of cell wall degrading enzymes is important for their modeling and industrial applications, including in the production of biofuels. Here we used Cel9A, a processive endocellulase from Thermobifida fusca, to demonstrate that cellulases that contain a catalytic domain (CD) attached to a cellulose binding module (CBM) by a flex...

Cellulases are key enzymes used in many processes for producing liquid fuels from biomass. Currently there many efforts to reduce the cost of cellulases using both structural approaches to improve the properties of individual cellulases and genomic approaches to identify new cellulases as well as other proteins that increase the activity of cellula...

Unlabelled: In nature, the complex composition and structure of the plant cell wall pose a barrier to enzymatic degradation. Nevertheless, some anaerobic bacteria have evolved for this purpose an intriguing, highly efficient multienzyme complex, the cellulosome, which contains numerous cellulases and hemicellulases. The rod-like cellulose componen...

The catalytic base in family 48 glycosyl hydrolases has not been previously established experimentally. Based on structural and modeling data published to date, we used site-directed mutagenesis and azide rescue activity assays to show definitively that the catalytic base in Thermobifida fusca Cel48A is aspartic acid 225. Of the tested mutants, onl...

Enzymatic hydrolysis of cellulose by microorganisms is a key step in the global carbon cycle. Despite its abundance only a small percentage of microorganisms can degrade cellulose, probably because it is present in recalcitrant cell walls. There are at least five distinct mechanisms used by different microorganisms to degrade cellulose all of which...

Designer cellulosomes are precision-engineered multienzyme complexes in which the molecular architecture and enzyme content are exquisitely controlled. This system was used to examine enzyme cooperation for improved synergy among Thermobifida fusca glycoside hydrolases. Two T. fusca cellulases, Cel48A exoglucanase and Cel5A endoglucanase, and two T...

Recent studies have shown that a number of glycoside hydrolase families do not follow the classical catalytic mechanisms, as they lack a typical catalytic base/nucleophile. A variety of mechanisms are used to replace this function, including substrate-assisted catalysis, a network of several residues, and the use of non-carboxylate residues or exog...

Anaerobic and aerobic bacteria use different mechanisms to degrade cellulose, with many aerobic microorganisms secreting a synergistic set of individual enzymes that includes multiple cellulases, most of which contain a carbohydrate-binding module (CBM) in addition to their catalytic domain (free enzyme mechanism). Anaerobic microorganisms usually...

The importance of cellulases in the production of fuels from biomass makes understanding their catalytic mechanisms on crystalline cellulose important in order to design more active enzymes. Seven modular cellulases from Thermobifida fusca have been purified and characterized; of which, three inverting cellulases: endocellulase Cel6A, exocellulase...

Cellulosomes are efficient cellulose-degradation systems produced by selected anaerobic bacteria. This multi-enzyme complex is assembled from a group of cellulases attached to a protein scaffold termed scaffoldin, mediated by a high-affinity protein-protein interaction between the enzyme-borne dockerin module and the cohesin module of the scaffoldi...

Conversion of components of the Thermobifida fusca free-enzyme system to the cellulosomal mode using the designer cellulosome approach can be employed to discover the properties and inherent advantages of the cellulosome system. In this article, we describe the conversion of the T. fusca xylanases Xyn11A and Xyn10B and their synergistic interaction...

Protein molecular scaffolds are attracting interest as natural candidates for the presentation of enzymes and acceleration of catalytic reactions. We have previously reported evidence that the stable protein 1 (SP1) from Populustremula can be employed as a molecular scaffold for the presentation of either catalytic or structural binding (cellulosom...

The shape and compaction of the bacterial nucleoid may affect the accessibility of genetic material to the transcriptional machinery in natural and synthetic systems. To investigate this phenomenon, the nature and contribution of RNA and protein to the compaction of nucleoids that had been gently released from Escherichia coli cells were investigat...

Amino acid modifications of the Thermobifida fusca Cel9A-68 catalytic domain or carbohydrate binding module 3c (CBM3c) were combined to create enzymes with changed amino acids in both domains. Bacterial crystalline cellulose (BC) and swollen cellulose (SWC) assays of the expressed and purified enzymes showed that three combinations resulted in 150%...

A relationship between processivity and synergism has not been reported for cellulases, although both characteristics are very important for hydrolysis of insoluble substrates. Mutation of two residues located in the active site tunnel of Thermobifida fusca exocellulase Cel6B increased processivity on filter paper. Surprisingly, mixtures of the Cel...

We have been developing the cellulases of Thermobifida fusca as a model to explore the conversion from a free cellulase system to the cellulosomal mode. Three of the six T. fusca cellulases (endoglucanase Cel6A and exoglucanases Cel6B and Cel48A) have been converted in previous work by replacing their cellulose-binding modules (CBMs) with a dockeri...

Summary Cellulose is the most abundant carbon source in nature but it is very difficult to degrade because of its insolubility, quasi-crystalline structure and its presence in plant cell walls in a matrix with other polymers that limit access to the cellulose surface. Most cellulose in soils is degraded by cellulolytic microorganisms that use a num...

There are two well studied mechanisms that are used by cellulolytic microorganisms to degrade the cellulose present in plant cell walls and a third less well studied oxidative mechanism used by brown rot fungi. The well studied mechanisms use cellulases to hydrolyze the β-1,4 linkages present in cellulose, however the way in which cellulases are pr...

There is a major international effort to develop renewable alternatives to fossil fuels. One approach is to produce a liquid fuel by enzymatically hydrolyzing carbohydrate polymers in biomass to sugars and fermenting them to ethanol. Cellulose is the main polymer in biomass and cellulases can hydrolyze it to cellobiose, which can be converted to gl...

Thermobifida fusca exocellulase Cel6B acts by an inverting hydrolysis mechanism; however, the catalytic acid and base residues for this enzyme have not been confirmed. Site-directed mutagenesis and kinetic studies were used to show that Asp274 is the catalytic acid, which is consistent with what is found for other members of family-6 glycoside hydr...

Thermobifida fuscaLam81A is a single domain family-81 beta-1,3-endoglucanase, but no structure is known for this family. Site-directed mutagenesis of 14 conserved residues chosen from sequence alignments was used to identify those with critical roles in catalysis, binding or substrate specificity. Mutant enzymes were assayed for their ability to bi...

We propose that transcript levels for some genes are affected by the bacterial cell division cycle and this may be an important factor to consider when designing synthetic bacterial genomes. To test this hypothesis, transcript levels of 58 genes in Escherichia coli B/r A were determined at five times during the cell division cycle. A two-step ANOVA...

E7, a single domain Family 33 cellulose binding module (CBM) protein, and E8, a non-catalytic, three-domain protein consisting of a Family 33 CBM, a FNIII domain, followed by a Family 2 CBM, were cloned, expressed, purified, and characterized. Western blots showed that E7 and E8 were induced and secreted when Thermobifida fusca was grown on cellobi...

Cellulose is a linear homopolymer of beta 1-4 linked glucose residues. Chitin is similar to cellulose in structure, and can be described as cellulose with the hydroxyl group on the C2 carbon replaced by an acetylamine group. Both cellulose and chitin form tightly packed, extensively hydrogen-bonded micro-fibrils. Up to now, binding of cellulase cat...

The synergistic action between Thermobifida fusca exocellulase Cel6B and endocellulase Cel5A on sodium hydroxide pretreated bacterial cellulose (BC) was determined. The activities of Cel6B and Cel5A were tested singly and both activities were dramatically increased on pretreated BC, especially in the early stage of hydrolysis. Cel5A, which attacks...

Cellulosomes are multi-enzyme complexes produced by certain anaerobic bacteria that exhibit efficient degradation of plant cell wall polysaccharides. To understand their enhanced levels of hydrolysis, we are investigating the effects of converting a free-cellulase system into a cellulosomal one. To achieve this end, we are replacing the cellulose-b...

1H NMR spectroscopy has been used to analyze the product profiles arising from the hydrolysis of cellooligosaccharides by family GH9 cellulases. The product profiles obtained with the wild type and several active site mutants of a bacterial processive endoglucanase, TfCel9A, were compared with those obtained by a randomly acting plant endoglucanase...

Cellulolytic bacteria and fungi have been shown to use two different approaches to degrade cellulose. Most aerobic microbes secrete sets of individual cellulases, many of which contain a carbohydrate binding molecule (CBM), which act synergistically on native cellulose. Most anaerobic microorganisms produce large multienzyme complexes called cellul...

CbhA and CelK are family-9 glycosyl hydrolases (β-1,4-glucanase) isolated from the bacterium Clostridium thermocellum. 1 The members of the glycosyl hydrolase (GH) family-9 have been almost exclusively endocellulolytic, with a few being processive endoglucanses (enzymes demonstrating both endo-and exocellulolytic activity). 2 CbhA and CelK are the...

The use of nLC-ESI-MS/MS in shotgun proteomics experiments and GeLC-MS/MS analysis is well accepted and routinely available in most proteomics laboratories. However, the same cannot be said for nLC-MALDI MS/MS, which has yet to experience such widespread acceptance, despite the fact that the MALDI technology offers several critical advantages over...

Self assembly is a prerequisite for fabricating nanoscale structures. Here we present a new fusion protein based on the stress-responsive homo-oligomeric protein, SP1. This ring-shaped protein is a highly stable homododecamer, which can be potentially utilized to self-assemble different modules and enzymes in a predicted and oriented manner. For th...

An inexpensive source of active cellulases is critical to efficient and cost-effective conversion of lignocellulosic biomass to ethanol. Transgenic plants expressing foreign cellulases are potential sources of cellulases for biomass conversion. A number of foreign proteins have been reported to accumulate to high levels when the transgene is incorp...

Thermobifida fusca secretes proteins that carry out plant cell wall degradation. Using two-dimensional electrophoresis, the extracellular proteome of T. fusca grown on cellobiose was compared to that of cells grown on glucose. Extracellular proteins, the expression of which is induced by cellobiose, mainly are cellulases and cellulose-binding prote...

In this study we employed Size Exclusion Chromatography (SEC) and X-ray diffraction to monitor the molecular weight and crystallinity of bacterial cellulose I and II (BC-I, BC-II) and microcrystalline cellulose (MCC) digested with three “pure” Thermobifida fusca cellulases (Cel6A, Cel6B, and Cel9A ). For each enzyme, cellulose crystallinity was fou...

The complete DNA sequence of the aerobic cellulolytic soil bacterium Cytophaga hutchinsonii, which belongs to the phylum Bacteroidetes, is presented. The genome consists of a single, circular, 4.43-Mb chromosome containing 3,790 open reading frames, 1,986 of which have been assigned a tentative function. Two of the most striking characteristics of...

Thermobifida fusca Cel9A-90 is a processive endoglucanase consisting of a family 9 catalytic domain (CD), a family 3c cellulose binding module (CBM3c), a fibronectin III-like domain, and a family 2 CBM. This enzyme has the highest activity of any individual T. fusca enzyme on crystalline substrates, particularly bacterial cellulose (BC). Mutations...

A critical structural feature of many microbial endo-β-1,4-glucanases (EGases, or cellulases) is a carbohydrate binding module (CBM), which is required for effective crystalline cellulose degradation. However, CBMs are absent from plant EGases that have been biochemically characterized to date, and accordingly, plant EGases are not generally though...

Fibrobacter succinogenes is one of the most active cellulolytic bacteria ever isolated from the rumen, but enzymes from F. succinogenes capable of hydrolyzing native (insoluble) cellulose at a rapid rate have not been identified. However, the genome sequence of F. succinogenes is now available, and it was hoped that this information would yield new...

Thermobifida fusca is a moderately thermophilic soil bacterium that belongs to Actinobacteria. It is a major degrader of plant cell walls and has been used as a model organism for the study of secreted, thermostable cellulases. The complete genome sequence showed that T. fusca has a single circular chromosome of 3,642,249 bp predicted to encode 3,1...

Cloning, high-level expression, and characterization of the somatotropin (ST) gene of an indigenous Nili-Ravi breed of water buffalo Bubalus bubalis (BbST) are described. Coding, non-coding, and promoter regions of BbST were amplified and sequenced. Sequence analysis revealed several silent and two interesting point mutations on comparison with STs...

Thermobifida fusca is a filamentous soil bacterium that plays a major role in the breakdown of plant biomass. In this paper, we report the cloning, expression, purification, and characterization of the T. fusca enzyme, Lam81A. The Carbohydrate Active Enzymes Database (http://afmb.cnrs-mrs.fr/CAZY/) indicates that Lam81A belongs to a relatively unch...