David. B. Wilson

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 importance for these microorganisms, GH48s have intrinsically low cellulolytic activity but even in low ratios synergize greatly with GH9 endoglucanases. In this study, we explore the structural and enzymatic diversity of these enzymes across a wide range of temperature optima. We have crystallized one new GH48 module from Bacillus pumilus in a complex with cellobiose and cellohexaose (BpumGH48). We compare this structure to other known GH48 enzymes in an attempt to understand GH48 structure/function relationships and draw general rules correlating amino acid sequences and secondary structures to thermostability in this GH family.

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 its active site, and explore the importance of the various domains of Thermobifida fusca AA10B (TfAA10B). The structure of TfAA10A is similar to other bacterial LPMOs (lytic polysaccharide monooxygenases), including signs of photo-reduction and a distorted active site, with mixed features showing both type I and II copper coordination. The point mutation experiments of TfAA10A show that Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for the binding of substrate, but that the X1 module does not affect binding or activity. In TfAA10A, Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for substrate binding, but that the X1 module does not affect binding or activity. The structure of TfAA10A is similar to other bacterial lytic polysaccharide monooxygenases with mixed features showing both type I and II copper coordination. The role of LPMOs and the variability of abundance in genomes are not fully explored. LPMOs likely perform initial attacks into crystalline cellulose to allow larger processive cellulases to bind and attack, but the precise nature of their synergistic behavior remains to be definitively characterized.

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 site in a continuous stream to participate in the processive reactions. Theoretical mutants of this enzyme were created in which all of the residues lining the pore were made hydrophobic, which had the effect in molecular dynamics simulations of emptying the pore of water molecules and preventing any from passing through the pore on the simulation time scale. Mutants with smaller numbers of substitutions of this nature, which could be created experimentally by site-directed mutagenesis, were also identified from simulations, and these proteins were subsequently produced in Escherichia coli, expressed and purified, but were found to not fold in a manner similar to the wild type protein, preventing the determination of the importance of the water pore for activity. It is possible that the presence of a small vacuum in the pore was responsible for the instability of the mutants. In addition, alternate pathways were observed in the simulations that would allow water molecules to reach the active site of the enzyme, suggesting that the hypothesis that the pore has functional significance might be incorrect.

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. Cel9A and Cel48A from a model bacterium, Thermobifida fusca (TfCel9A and TfCel48A, respectively), are two cellulases with different properties and have previously been shown to synergize well with each other. TfCel9A is a processive endocellulase with relatively high activity on crystalline cellulose. TfCel48A is a reducing end-directed exocellulase with very low activity on crystalline cellulose. Neither enzyme fits its respective role in the classical synergism model of enzymatic cellulose digestion. Using the results of time course, endpoint, and sequential addition activity assays, we propose a model of synergistic cooperation between the two cellulases. TfCel9A is most effective on fresh bacterial cellulose with a presumably uniform surface at the molecular level. Its processive activity likely erodes the surface and thus reduces its own activity. TfCel48A is able to hydrolyze the TfCel9A-modified substrate efficiently and replenish the uniform surface required by TfCel9A, creating a feedback mechanism. The model of synergistic interactions is comparable to an earlier proposed model for Trichoderma reesei Cel7A and Cel7B, but the roles of endo- and exocellulases are reversed, a finding which suggests that bacteria and fungi may have evolved different approaches to efficient biomass degradation.

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 strategies are commonly observed either separately or in combination. These include free enzyme systems, multifunctional enzymes, and multi-enzyme self-assembled designer cellulosome complexes. Results: In order to compare these different paradigms, we employed a synthetic biology approach to convert two different cellulases from the free enzymatic system of the well-studied bacterium, Thermobifida fusca, into bifunctional enzymes with different modular architectures. We then examined their performance compared to those of the combined parental free-enzyme and equivalent designer-cellulosome systems. The results showed that the cellulolytic activity displayed by the different architectures of the bifunctional enzymes was somewhat inferior to that of the wild-type free enzyme system. Conclusions: The activity exhibited by the designer cellulosome system was equal or superior to that of the free system, presumably reflecting the combined proximity of the enzymes and high flexibility of the designer cellulosome components, thus enabling efficient enzymatic activity of the catalytic modules.

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 of which have their active sites in a tunnel, couple movement along a cellulose chain with cleavage of cellobiose from the end of the cellulose molecule. There are two sets of structures that suggest how an exocellulase might move along a cellulose chain. For family 48 exocellulases there are two different ways that a chain can be bound in the active site while for family 6 exocellulases there are several different ligand-bound structures. Site-directed mutagenesis of Thermobifida fusca exocellulases Cel48A and Cel6B and the processive endoglucanase Cel9A have identified some mutations that increase processivity and some that decrease processivity. In addition a mutation in Cel6B was identified that appears to allow the mutant enzyme to move along a cellulose chain in the absence of cleavage.

(2012). Synergistic interactions in cellulose hydrolysis. Biofuels: Vol. 3, No. 1, pp. 61-70.

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 cellulases in degrading pretreated biomass materials. Fungal GH-61 proteins are important new enzymes that increase the activity of current commercial cellulases leading to lower total protein loading and thus lower cost. Recent work has greatly increased our knowledge of these novel enzymes that appear to be oxido-reductases that target crystalline cellulose and increase its accessibility to cellulases. They appear to carry out the C1 activity originally proposed by Dr Reese. Cellobiose dehydrogenase appears to interact with GH-61 proteins in this function, providing a role for this puzzling enzyme. Cellulase research is making considerable progress and appears to be poised for even greater advances.

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, only Cel48A with the D225E mutation retained partial activity on soluble and insoluble substrates. In azide rescue experiments, only the D225G mutation, in the smallest residue tested, showed an increase in activity with added azide.