Clostridium Thermocellum Cellulosome Research Articles

Immobilization of Recombinant Endoglucanase (CelA) from Clostridium thermocellum on Modified Regenerated Cellulose Membrane

Cellulases are being widely employed in lignocellulosic biorefineries for the sustainable production of value-added bioproducts. However, the high production cost, sensitivity, and non-reusability of free cellulase enzymes impede their commercial applications. Enzyme immobilization seems to be a potential approach to address the aforesaid complications. The current study aims at the production of recombinant endoglucanase (CelA) originated from the cellulosome of Clostridium thermocellum in Escherichia coli (E. coli), followed by immobilization using modified regenerated cellulose (RC) membranes. The surface modification of RC membranes was performed in two different ways: one to generate the immobilized metal ion affinity membranes RC-EPI-IDA-Co2+ (IMAMs) for coordination coupling and another to develop aldehyde functional group membranes RC-EPI-DA-GA (AMs) for covalent bonding. For the preparation of IMAMs, cobalt ions expressed the highest affinity effect compared to other metal ions. Both enzyme-immobilized membranes exhibited better thermal stability and maintained an improved relative activity at higher temperatures (50–90 °C). In the storage analysis, 80% relative activity was retained after 15 days at 4 °C. Furthermore, the IMAM- and AM-immobilized CelA retained 63% and 53% relative activity, respectively, after being reused five times. As to the purification effect during immobilization, IMAMs showed a better purification fold of 3.19 than AMs. The IMAMs also displayed better kinetic parameters, with a higher Vmax of 15.57 U mg−1 and a lower Km of 36.14 mg mL−1, than those of AMs. The IMAMs were regenerated via treatment with stripping buffer and reloaded with enzymes and displayed almost 100% activity, the same as free enzymes, up to 5 cycles of regeneration.

Directed Evolution of Clostridium thermocellum β-Glucosidase A Towards Enhanced Thermostability.

β-Glucosidases are key enzymes in the process of cellulose utilization. It is the last enzyme in the cellulose hydrolysis chain, which converts cellobiose to glucose. Since cellobiose is known to have a feedback inhibitory effect on a variety of cellulases, β-glucosidase can prevent this inhibition by hydrolyzing cellobiose to non-inhibitory glucose. While the optimal temperature of the Clostridium thermocellum cellulosome is 70 °C, C. thermocellum β-glucosidase A is almost inactive at such high temperatures. Thus, in the current study, a random mutagenesis directed evolutionary approach was conducted to produce a thermostable mutant with Kcat and Km, similar to those of the wild-type enzyme. The resultant mutant contained two mutations, A17S and K268N, but only the former was found to affect thermostability, whereby the inflection temperature (Ti) was increased by 6.4 °C. A17 is located near the central cavity of the native enzyme. Interestingly, multiple alignments revealed that position 17 is relatively conserved, whereby alanine is replaced only by serine. Upon the addition of the thermostable mutant to the C. thermocellum secretome for subsequent hydrolysis of microcrystalline cellulose at 70 °C, a higher soluble glucose yield (243%) was obtained compared to the activity of the secretome supplemented with the wild-type enzyme.

Effect of expression of additional catalytic domain on characteristics of Xylanase Z of Clostridium thermocellum

Xylanase Z (XynZ) is a prime part of cellulosome of Clostridium thermocellum. The several domains of the cellulosome complexes are frequently tethered to one another by intrinsically messy regions. This is multidomain xylanase comprising of N-terminus feruloyl esterase followed by substrate binding, dockerin and the catalytic domains. This study design was established to check the expression, activities and other characteristics of different variants of Xylanase Z produced by the C-terminal (XynZ-CB), N-terminal (XynZ-BC) and both terminals (XynZ-BCB) binding domain to catalytic domain in different arrangements. XynZ variants of C. thermocellum with binding domain committed with catalytic domain of C-terminal (XynZ-CB) and N-terminal (XynZ-BC) and both terminals (XynZ-BCB) binding domain and one with catalytic domain at both terminals of the binding domain (XynZ-CBC) were checked in E. coli in different levels like 15, 30, 30, and 33% of cell proteins, respectively. The specific xylanase Z activities of XynZ-CB, XynZ BC, XynZ-BCB and XynZ-BCB were 72, 68, 67 and 200 U mg−1, though their equimolar basis activities were 4176, 3944, 5092 and 19,200 U μM−1 compared to xylan (birchwood). The over-all activities of XynZ-CB, XynZ-BC, XynZ-BCB and XynZ-CBC produced in fermentation culture were 3678, 3579, 3800 and 5400 U L−1 OD600−1, respectively. The results showed that overall yield of XynZ-C remained manifold higher than all other variants due to combined effect of its specific activity and expression level while the activity of variant XynZ-CBC was increased four folds on equimolar basis as compared to its native form. The fruit saccharification increased with the treatment of xylanase and cellulase.

Dynamic interactions of type I cohesin modules fine-tune the structure of the cellulosome of Clostridium thermocellum

Efficient degradation of plant cell walls by selected anaerobic bacteria is performed by large extracellular multienzyme complexes termed cellulosomes. The spatial arrangement within the cellulosome is organized by a protein called scaffoldin, which recruits the cellulolytic subunits through interactions between cohesin modules on the scaffoldin and dockerin modules on the enzymes. Although many structural studies of the individual components of cellulosomal scaffoldins have been performed, the role of interactions between individual cohesin modules and the flexible linker regions between them are still not entirely understood. Here, we report single-molecule measurements using FRET to study the conformational dynamics of a bimodular cohesin segment of the scaffoldin protein CipA of Clostridium thermocellum We observe compacted structures in solution that persist on the timescale of milliseconds. The compacted conformation is found to be in dynamic equilibrium with an extended state that shows distance fluctuations on the microsecond timescale. Shortening of the intercohesin linker does not destabilize the interactions but reduces the rate of contact formation. Upon addition of dockerin-containing enzymes, an extension of the flexible state is observed, but the cohesin-cohesin interactions persist. Using all-atom molecular-dynamics simulations of the system, we further identify possible intercohesin binding modes. Beyond the view of scaffoldin as "beads on a string," we propose that cohesin-cohesin interactions are an important factor for the precise spatial arrangement of the enzymatic subunits in the cellulosome that leads to the high catalytic synergy in these assemblies and should be considered when designing cellulosomes for industrial applications.

Novel insights into the degradation of β-1,3-glucans by the cellulosome of Clostridium thermocellum revealed by structure and function studies of a family 81 glycoside hydrolase

The family 81 glycoside hydrolase (GH81) from Clostridium thermocellum is a β-1,3-glucanase belonging to cellulosomal complex. The gene encoding GH81 from Clostridium thermocellum (CtLam81A) was cloned and expressed displaying a molecular mass of ~82 kDa. CtLam81A showed maximum activity against laminarin (100 U/mg), followed by curdlan (65 U/mg), at pH 7.0 and 75 °C. CtLam81A displayed Km, 2.1 ± 0.12 mg/ml and Vmax, 109 ± 1.8 U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca2+ or Mg2+ ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 °C to 96 °C by Ca2+ or Mg2+ ions and decreased to 82 °C by EDTA, indicating that Ca2+ and Mg2+ ions may be involved in catalysis and in maintaining structural integrity. TLC and MALDI-TOF analysis of β-1,3-glucan hydrolysed products released initially, showed β-1,3-glucan-oligosaccharides degree of polymerization (DP) from DP2 to DP7, confirming an endo-mode of action. The catalytically inactive mutant CtLam81A-E515A generated by site-directed mutagenesis was co-crystallized and tetragonal crystals diffracting up to 1.4 Å resolution were obtained. CtLam81A-E515A contained 15 α-helices and 38 β-strands forming a four-domain structure viz. a β-sandwich domain I at N-terminal, an α/β-domain II, an (α/α)6 barrel domain III, and a small 5-stranded β-sandwich domain IV.

Steered molecular dynamics simulations reveal the role of Ca2+ in regulating mechanostability of cellulose-binding proteins.

The conversion of cellulosic biomass into biofuels requires degradation of the biomass into fermentable sugars. The most efficient natural cellulase system for carrying out this conversion is an extracellular multi-enzymatic complex named the cellulosome. In addition to temperature and pH stability, mechanical stability is important for functioning of cellulosome domains, and experimental techniques such as Single Molecule Force Spectroscopy (SMFS) have been used to measure the mechanical strength of several cellulosomal proteins. Molecular dynamics computer simulations provide complementary atomic-resolution quantitative maps of domain mechanical stability for identification of experimental leads for protein stabilization. In this study, we used multi-scale steered molecular dynamics computer simulations, benchmarked against new SMFS measurements, to measure the intermolecular contacts that confer high mechanical stability to a family 3 Carbohydrate Binding Module protein (CBM3) derived from the archetypal Clostridium thermocellum cellulosome. Our data predicts that electrostatic interactions in the calcium binding pocket modulate the mechanostability of the cellulose-binding module, which provides an additional design rule for the rational re-engineering of designer cellulosomes for biotechnology. Our data offers new molecular insights into the origins of mechanostability in cellulose binding domains and gives leads for synthesis of more robust cellulose-binding protein modules. On the other hand, simulations predict that insertion of a flexible strand can promote alternative unfolding pathways and dramatically reduce the mechanostability of the carbohydrate binding module, which gives routes to rational design of tailormade fingerprint complexes for force spectroscopy experiments.

Hydrolysis of model cellulose films by cellulosomes: Extension of quartz crystal microbalance technique to multienzymatic complexes

Bacterial cellulosomes contain highly efficient complexed cellulases and have been studied extensively for the production of lignocellulosic biofuels and bioproducts. A surface measurement technique, quartz crystal microbalance with dissipation (QCM-D), was extended for the investigation of real-time binding and hydrolysis of model cellulose surfaces from free fungal cellulases to the cellulosomes of Clostridium thermocellum (Ruminiclostridium thermocellum). In differentiating the activities of cell-free and cell-bound cellulosomes, greater than 68% of the cellulosomes in the crude cell broth were found to exist unattached to the cell across multiple growth stages. The initial hydrolysis rate of crude cell broth measured by QCM was greater than that of cell-free cellulosomes, but the corresponding frequency drop (a direct measure of the mass of enzyme adsorbed to the film) of crude cell broth was less than that of the cell-free cellulosomes, consistent with the underestimation of the cell mass adsorbed using QCM. Inhibition of hydrolysis by cellobiose (0–10g/L), which is similar for crude cell broth and cell-free cellulosomes, demonstrates the sensitivity of the QCM to environmental perturbations of multienzymatic complexes. QCM measurements using multienzymatic complexes may be used to screen and optimize hydrolysis conditions and to develop mechanistic, surface-based models of enzymatic cellulose deconstruction.

Enzymatic diversity of the Clostridium thermocellum cellulosome is crucial for the degradation of crystalline cellulose and plant biomass.

The cellulosome is a supramolecular multienzyme complex comprised of a wide variety of polysaccharide-degrading enzymes and scaffold proteins. The cellulosomal enzymes that bind to the scaffold proteins synergistically degrade crystalline cellulose. Here, we report in vitro reconstitution of the Clostridium thermocellum cellulosome from 40 cellulosomal components and the full-length scaffoldin protein that binds to nine enzyme molecules. These components were each synthesized using a wheat germ cell-free protein synthesis system and purified. Cellulosome complexes were reconstituted from 3, 12, 30, and 40 components based on their contents in the native cellulosome. The activity of the enzyme-saturated complex indicated that greater enzymatic variety generated more synergy for the degradation of crystalline cellulose and delignified rice straw. Surprisingly, a less complete enzyme complex displaying fewer than nine enzyme molecules was more efficient for the degradation of delignified rice straw than the enzyme-saturated complex, despite the fact that the enzyme-saturated complex exhibited maximum synergy for the degradation of crystalline cellulose. These results suggest that greater enzymatic diversity of the cellulosome is crucial for the degradation of crystalline cellulose and plant biomass, and that efficient degradation of different substrates by the cellulosome requires not only a different enzymatic composition, but also different cellulosome structures.

The Contribution of Non-catalytic Carbohydrate Binding Modules to the Activity of Lytic Polysaccharide Monooxygenases

Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharide monooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases, CfLPMO10 and TbLPMO10 from Cellulomonas fimi and Thermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducing CtCBM3a, from the Clostridium thermocellum cellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact of CtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM from CfLPMO10 or the introduction of a family 10 CBM from Cellvibrio japonicus LPMO10B into TbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations.

Stiffness of the C-terminal disordered linker affects the geometry of the active site in endoglucanase Cel8A.

Cellulosomes are complex multi-enzyme machineries which efficiently degrade plant cell-wall polysaccharides. The multiple domains of the cellulosome proteins are often tethered together by intrinsically disordered regions. The properties and functions of these disordered linkers are not well understood. In this work, we study endoglucanase Cel8A, which is a relevant enzymatic component of the cellulosomes of Clostridium thermocellum. We use both all-atom and coarse-grained simulations to investigate how the conformations of the catalytic domain of Cel8A are affected by the disordered linker at its C terminus. We find that when the endoglucanase is bound to its substrate, the effective stiffness of the linker can influence the distances between groups of amino-acid residues throughout the entire enzymatic domain. In particular, variations in the linker stiffness can lead to small changes in the geometry of the active-site cleft. We suggest that such geometrical changes may have an effect on the catalytic activity of the enzyme.

Cellulose hydrolysis ability of a Clostridium thermocellum cellulosome containing small-size scaffolding protein CipA

Mutant Clostridium thermocellum YM72 that produces small-size scaffolding protein CipA (ssCipA) was isolated from wild-type YM4. Sequencing of ssCipA revealed that two domains, cohesin 6 and cohesin 7, were not present. Cellulosome prepared from YM72 exhibited a significant reduction of hydrolysis ability on crystalline celluloses such as Sigmacell type-20 and cellulose from Halocynthia. To investigate this influence in vitro, artificial cellulosomes were assembled as recombinant CipA (rCipA) and ssCipA (rssCipA) using native free-cellulosomal subunits. The cellulosome assembled using rssCipA showed a 1.8-fold decrease in the hydrolysis of crystalline cellulose compared with that of rCipA. However, no significant differences in the hydrolysis of carboxymethylcellulose and acid-swollen cellulose were observed. One protein band was missing from the complex that was assembled using rssCipA (confirmed by native-PAGE). The missing protein was identified as CelJ, which is a major cellulosomal subunit. This suggests that insufficient cooperation of CelJ into the cellulosome results in the significant reduction of hydrolysis toward crystalline cellulose. These results indicate that cohesin 6 and 7 may be responsible for the cooperation of CelJ through cohesin and dockerin interactions, and adequate cooperation of CelJ into the cellulosome is important for significant hydrolysis of crystalline cellulose.

Statistical radii associated with amino acids to determine the contact map: fixing the structure of a type I cohesin domain in the Clostridium thermocellum cellulosome

We propose to improve and simplify protein refinement procedures through consideration of which pairs of amino acid residues should form native contacts. We first consider 11 330 proteins from the CATH database to determine statistical distributions of contacts associated with a given type of amino acid. The distributions are set across the distances between the α-C atoms that are in contact. Based on this data, we determine typical radii of effective spheres that can be placed on the α-C atoms in order to reconstruct the distribution of the contact lengths. This is done by checking for overlaps with enlarged van der Waals spheres associated with heavy atoms on other amino acids.The resulting contacts can be used to identify non-native contacts that may arise during the time evolution of structure-based models. Here, the radii are used to guide reconstruction of nine missing side chains in a type I cohesin domain with the Protein Data Bank code 1AOH. We first identify the likely missing contacts and then sculpt the corresponding side chains by standard refinement tools to achieve consistency with the expected contact map. One ambiguity in refinement is resolved by determining all-atom conformational energies.

Stoichiometric Assembly of the Cellulosome Generates Maximum Synergy for the Degradation of Crystalline Cellulose, as Revealed by In Vitro Reconstitution of the Clostridium thermocellum Cellulosome.

The cellulosome is a supramolecular multienzyme complex formed by species-specific interactions between the cohesin modules of scaffoldin proteins and the dockerin modules of a wide variety of polysaccharide-degrading enzymes. Cellulosomal enzymes bound to the scaffoldin protein act synergistically to degrade crystalline cellulose. However, there have been few attempts to reconstitute intact cellulosomes due to the difficulty of heterologously expressing full-length scaffoldin proteins. We describe the synthesis of a full-length scaffoldin protein containing nine cohesin modules, CipA; its deletion derivative containing two cohesin modules, ΔCipA; and three major cellulosomal cellulases, Cel48S, Cel8A, and Cel9K, of the Clostridium thermocellum cellulosome. The proteins were synthesized using a wheat germ cell-free protein synthesis system, and the purified proteins were used to reconstitute cellulosomes. Analysis of the cellulosome assembly using size exclusion chromatography suggested that the dockerin module of the enzymes stoichiometrically bound to the cohesin modules of the scaffoldin protein. The activity profile of the reconstituted cellulosomes indicated that cellulosomes assembled at a CipA/enzyme molar ratio of 1/9 (cohesin/dockerin = 1/1) and showed maximum synergy (4-fold synergy) for the degradation of crystalline substrate and ∼2.4-fold-higher synergy for its degradation than minicellulosomes assembled at a ΔCipA/enzyme molar ratio of 1/2 (cohesin/dockerin = 1/1). These results suggest that the binding of more enzyme molecules on a single scaffoldin protein results in higher synergy for the degradation of crystalline cellulose and that the stoichiometric assembly of the cellulosome, without excess or insufficient enzyme, is crucial for generating maximum synergy for the degradation of crystalline cellulose.

A kinetics modeling study on the inhibition of glucose on cellulosome of Clostridium thermocellum

A simplified kinetics model was built to study the inhibition of glucose on cellulosome of Clostridium thermocellum. Suitable reaction conditions were adopted to evaluate the model. The model was evaluated at different temperatures and further with various activated carbon additions as adsorbent for glucose. Investigation results revealed that the model could describe the hydrolysis kinetics of cellulose by cellulosome quite well. Glucose was found to be an inhibitor for cellulosome based on the kinetics analysis. Inhibition increased with the increase in temperature. Activated carbon as adsorbent could lower the inhibition. Parameters in the model were further discussed based on the experiment. The model might also be used to describe the strong inhibition of cellobiose on cellulosome. Saccharification of cellulose by both cellulosome and C. thermocellum could be enhanced efficiently by activated carbon addition.

Resonance assignments of the periplasmic domain of a cellulose-sensing trans-membrane anti-sigma factor from Clostridium thermocellum.

The cellulosome of Clostridium thermocellum is an elegant and efficient multi-enzyme complex for degrading lignocellulose. The cellulosome contains several dozens of carbohydrate hydrolysis enzymes, which are regulated by the presence of environmental substrates through several pairs of sigma and anti-sigma factors. The anti-sigma factors sense the presence of substrates and transduce the signals into the cell. The sigma factors are then released from the corresponding anti-sigma factors, and they recruit RNA polymerase to transcribe specific cellulosomal genes. However, it is not clear how the extracellular signals are transduced into the cell by the anti-sigma factors. The anti-sigma factors of C. thermocellum contain an N-terminal intracellular domain, a trans-membrane helix, a periplasmic domain, a proline-rich region which is probably required for crossing the cell wall, and a C-terminal carbohydrate-binding domain or glycoside hydrolase domain. The periplasmic domain may play a key role in signal transduction; however, its three-dimensional structure is still unknown. Here we report the NMR resonance assignments of the periplasmic domain of anti-sigma factor RsgI2 from C. thermocellum as a basis for further structural determination and functional studies.

Role of pectinolytic enzymes identified in Clostridium thermocellum cellulosome.

The cloning, expression and characterization of three cellulosomal pectinolytic enzymes viz., two variants of PL1 (PL1A and PL1B) and PL9 from Clostridium thermocellum was carried out. The comparison of the primary sequences of PL1A, PL1B and PL9 revealed that these proteins displayed considerable sequence similarities with family 1 and 9 polysaccharide lyases, respectively. PL1A, PL1B and PL9 are the putative catalytic domains of protein sequence ABN54148.1 and ABN53381.1 respectively. These two protein sequences also contain putative carbohydrate binding module (CBM) and type-I dockerin. The associated putative CBM of PL1A showed strong homology with family 6 CBMs while those of PL1B and PL9 showed homology with family 35 CBMs. Recombinant derivatives of these three enzymes showed molecular masses of approximately 34 kDa, 40 kDa and 32 kDa for PL1A, PL1B and PL9, respectively. PL1A, PL1B and PL9 displayed high activity toward polygalacturonic acid and pectin (up to 55% methyl-esterified) from citrus fruits. However, PL1B showed relatively higher activity towards 55% and 85% methyl-esterified pectin (citrus). PL1A and PL9 showed higher activity on rhamnogalacturonan than PL1B. Both PL1A and PL9 displayed maximum activity at pH 8.5 with optimum temperature of 50°C and 60°C respectively. PL1B achieved highest activity at pH 9.8, under an optimum temperature of 50°C. PL1A, PL1B and PL9 all produced two or more unsaturated galacturonates from pectic substrates as displayed by TLC analysis confirming that they are endo-pectate lyase belonging to family 1 and 9, respectively. This report reveals that pectinolytic activity displayed by Clostridium thermocellum cellulosome is coordinated by a sub-set of at least three multi-modular enzymes.

In silico structural characterization and molecular docking studies of first glucuronoxylan-xylanohydrolase (Xyn30A) from family 30 glycosyl hydrolase (GH30) from Clostridium thermocellum

CtXynGH30 is a carbohydrate active modular enzyme and component of cellulosome of Clostridium thermocellum. The full length CtXynGH30 contains an N-terminal catalytic module named as Xyn30A and a family 6 carbohydrate binding module (CBM6) at C-terminuis. Xyn30A was modeled by computer program Modeller9v8 taking crystal structure of XynC from B. subtilis as a template to generate the molecular model. Model refinement was done using energy minimization by implementing steepest descent algorithm with GROMOS96 43al force field. Quality assessment by Ramachandran plot showed that 91% amino acids lie in most favourable region and 9% in additional allowed region. Structural analysis depicted that Xyn30A has a (beta/alpha)8 barrel fold. Ad- ditionally, it had a beta-strand rich structure called 'side beta-structure' attached with main catalytic core. Structural superimposition reflected that Glu136 act as a catalytic acid/base while Glu225 act as a catalytic nucleophile. Multiple sequence alignment showed that these catalytic residues are conserved within the family. The docking results showed that these residues display polar interaction with linear and substituted xylo-oligosaccharides. The binding interaction of ligands depicted that aromatic amino acids Trp81, Tyr139, Trp143, Phe172, His198, Tyr200, Tyr227, Trp264 and Tyr265 create binding site pocket around the active site. We report overall structural feature, conserved active site residues and enzymeligand docking of first glucuronoxylan-xylanohydrolase (Xyn30A) of family 30 glycosyl hydrolase (GH30) from Clostridium thermocellum.

Structure of a family 3a carbohydrate-binding module from the cellulosomal scaffoldin CipA of Clostridium thermocellum with flanking linkers: implications for cellulosome structure.

The cellulosome of the cellulolytic bacterium Clostridium thermocellum has a structural multi-modular protein called CipA (cellulosome-integrating protein A) that includes nine enzyme-binding cohesin modules and a family 3 cellulose-binding module (CBM3a). In the CipA protein, the CBM3a module is located between the second and third cohesin modules and is connected to them via proline/threonine-rich linkers. The structure of CBM3a with portions of the C- and N-terminal flanking linker regions, CBM3a-L, has been determined to a resolution of 1.98 Å. The structure is a β-sandwich with a structural Ca(2+) ion. The structure is consistent with the previously determined CipA CBM structure; however, the structured linker regions provide a deeper insight into the overall cellulosome structure and assembly.

Cell-free protein synthesis and substrate specificity of full-length endoglucanase CelJ (Cel9D-Cel44A), the largest multi-enzyme subunit of theClostridium thermocellumcellulosome

Endoglucanase CelJ (Cel9D-Cel44A) is the largest multi-enzyme subunit of the Clostridium thermocellum cellulosome and is composed of glycoside hydrolase (GH) families 9 and 44 (GH9 and GH44) and carbohydrate-binding module (CBM) families 30 and 44 (CBM30 and CBM44). The study of CelJ has been hampered by the inability to isolate full-length CelJ from recombinant Escherichia coli cells. Here, full-length CelJ and its N- and C-terminal segments, CBM30-GH9 (Cel9D) and GH44-CBM44 (Cel44A), were synthesized using a wheat germ cell-free protein synthesis system and then were purified to homogeneity. Analysis of the substrate specificities of CelJ and its derivatives demonstrated that the fusion of Cel9D and Cel44A results in threefold synergy for the degradation of xyloglucan, one of the major structural polysaccharides of plant cell walls. Because CelJ displayed broad substrate specificity including significant carboxymethylcellulase (CMCase) and xylanase activities in addition to high xyloglucanase activity, CelJ may play an important role in the degradation of plant cell walls, which are composed of highly heterogeneous polysaccharides. Furthermore, because Cel9D, but not Cel44A, acts as a semi-processive endoglucanase, the different modes of action between Cel9D and Cel44A may be responsible for the observed synergistic effect on the activity of CelJ (Cel9D-Cel44A).

Small Angle X-ray Scattering Analysis of Clostridium thermocellum Cellulosome N-terminal Complexes Reveals a Highly Dynamic Structure

Clostridium thermocellum produces the prototypical cellulosome, a large multienzyme complex that efficiently hydrolyzes plant cell wall polysaccharides into fermentable sugars. This ability has garnered great interest in its potential application in biofuel production. The core non-catalytic scaffoldin subunit, CipA, bears nine type I cohesin modules that interact with the type I dockerin modules of secreted hydrolytic enzymes and promotes catalytic synergy. Because the large size and flexibility of the cellulosome preclude structural determination by traditional means, the structural basis of this synergy remains unclear. Small angle x-ray scattering has been successfully applied to the study of flexible proteins. Here, we used small angle x-ray scattering to determine the solution structure and to analyze the conformational flexibility of two overlapping N-terminal cellulosomal scaffoldin fragments comprising two type I cohesin modules and the cellulose-specific carbohydrate-binding module from CipA in complex with Cel8A cellulases. The pair distribution functions, ab initio envelopes, and rigid body models generated for these two complexes reveal extended structures. These two N-terminal cellulosomal fragments are highly dynamic and display no preference for extended or compact conformations. Overall, our work reveals structural and dynamic features of the N terminus of the CipA scaffoldin that may aid in cellulosome substrate recognition and binding.