cellulase and cellohexaose

Topics: Amino Acid Sequence; Bacillus; Bacterial Proteins; Biocatalysis; Cellulase; Endo-1,4-beta Xylanases; Molecular Docking Simulation; Multifunctional Enzymes; Mutagenesis, Site-Directed; Mutation; Oligosaccharides; Protein Binding; Protein Engineering; Xylans

Lignocellulosic biomass is anticipated to serve as a platform for green chemicals and fuels. Nonproductive binding of lignin to cellulolytic enzymes should be avoided for conversion of lignocellulose through enzymatic saccharification. Although carbohydrate-binding modules (CBMs) of cellulolytic enzymes strongly bind to lignin, the adsorption mechanism at molecular level is still unclear. Here, we report NMR-based analyses of binding sites on CBM1 of cellobiohydrolase I (Cel7A) from a hyper-cellulase-producing fungus, Trichoderma reesei, with cellohexaose and lignins from Japanese cedar (C-MWL) and Eucalyptus globulus (E-MWL). A method was established to obtain properly folded TrCBM1. Only TrCBM1 that was expressed in freshly transformed E. coli had intact conformation. Chemical shift perturbation analyses revealed that TrCBM1 adsorbed cellohexaose in highly specific manner via two subsites, flat plane surface and cleft, which were located on the opposite side of the protein surface. Importantly, MWLs were adsorbed at multiple binding sites, including the subsites, having higher affinity than cellohexaose. G6 and Q7 were involved in lignin binding on the flat plane surface of TrCBM1, while cellohexaose preferentially interacted with N29 and Q34. TrCBM1 used much larger surface area to bind with C-MWL than E-MWL, indicating the mechanisms of adsorption toward hardwood and softwood lignins are different.

Topics: Amino Acids; Binding Sites; Cedrus; Cellulase; Cellulose 1,4-beta-Cellobiosidase; Eucalyptus; Lignin; Oligosaccharides; Receptors, Cell Surface; Trichoderma

Optimal catalytic activity of endoglucanase Cel5D from the thermophilic anaerobic bacterium Caldicellulosiruptor bescii requires the presence of a carbohydrate-binding module of family 28, CbCBM28. The binding properties of CbСВМ28 with cello-, laminari-, xylo- and chito-oligosaccharides were studied by isothermal titration calorimetry. CbСВМ28 bound only cello-oligosaccharides comprising at least four glucose residues with binding constants of 2.5·10

Topics: Binding Sites; Calorimetry; Cellulase; Cellulose; Crystallins; Firmicutes; Glucose; Hydrogen-Ion Concentration; Oligosaccharides; Tetroses

Endo-ß-1,4-glucanase AkEG21 belonging to glycosyl hydrolase family 45 (GHF45) is the most abundant cellulase in the digestive fluid of sea hare (Aplysia kurodai). The specific activity of this 21-kDa enzyme is considerably lower than those of other endo ß-1,4-glucanases in the digestive fluid of A. kurodai, therefore its role in whole cellulose hydrolysis by sea hare is still uncertain. Although AkEG21 has a catalytic domain without a cellulose binding domain, it demonstrated stable binding to cellulose fibers, similar to that of fungal cellobiohydrolase (CBH) 1 and CBH 2, which is strongly inhibited by cellohexaose, suggesting the involvement of the catalytic site in cellulose binding. Cellulose-bound AkEG21 hydrolyzed cellulose to cellobiose, cellotriose and cellotetraose, but could not digest an external substrate, azo-carboxymethyl cellulose. Cellulose hydrolysis was considerably stimulated by the synergistic action of cellulose-bound AkEG21 and AkEG45, another ß-1,4-endoglucanase present in the digestive fluid of sea hare; however no synergy in carboxymethylcellulose hydrolysis was observed. When AkEG21 was removed from the digestive fluid by immunoprecipitation, the cellulose hydrolyzing activity of the fluid was significantly reduced, indicating a critical role of AkEG21 in cellulose hydrolysis by A. kurodai. These findings suggest that AkEG21 is a processive endoglucanase functionally equivalent to the CBH, which provides a CBH-independent mechanism for the mollusk to digest seaweed cellulose to glucose.

Topics: Animals; Aplysia; Catalytic Domain; Cellobiose; Cellulase; Cellulose; Digestion; Glucose; Hydrolysis; Kinetics; Oligosaccharides; Protein Binding; Protein Domains; Tetroses

Ionic liquids (ILs), that is, salts with melting points <100°C, have recently attracted a lot of attention in biomass processing due to their ability to dissolve lignocellulosics. In this work, we studied how two imidazolium-based, hydrophilic, cellulose dissolving ionic liquids 1,3-dimethylimidazolium dimethylphosphate [DMIM]DMP and 1-ethyl-3-methylimidazolium acetate [EMIM]AcO affect the usually employed analytical methods for mono- and oligosaccharides, typical products from hydrolytic treatments of biomass. HPLC methods were severely hampered by the presence of ILs with loss of separation power and severe baseline problems, making their use for saccharide quantification extremely challenging. Problems in DNS photometric assay and chromatography were also encountered at high ionic liquid concentrations and many capillary electrophoresis (CE) methods did not allow an efficient analysis of saccharides in these matrices. In this paper we describe an optimized CE method with pre-column derivatization for the qualitative and quantitative analysis of mono- and oligosaccharides in sample matrices containing moderate (20-40% (v/v)) concentrations of ILs. The IL content and type in the sample matrix was found to affect both peak shape and quantification parameters. Generally, the presence of high IL concentrations (≥20% (v/v)) had a dampening effect on the detection of the analytes. IL in lower concentrations of <20% (v/v) was, however, found to improve peak shape and/or separation in some cases. The optimized CE method has good sensitivity in moderate concentrations of the ionic liquids used, with limits of detection of 5mg/L for cellooligomers up to the size of cellotetraose and 5-20mg/L for cellopentaose and cellohexaose, depending on the matrix. The method was used for analysing the action of a commercial β-glucosidase in ILs and for analysing saccharides in the IL containing hydrolysates from the hydrolysis of microcrystalline cellulose with Trichoderma reesei endoglucanase Cel5A. According to the results, [DMIM]DMP and [EMIM]AcO] showed clear differences in enzyme inactivation.

Topics: Calibration; Cellulase; Cellulose; Electrolytes; Electrophoresis, Capillary; Imidazoles; Ionic Liquids; Oligosaccharides; Photometry; Tetroses; Trichoderma

Efforts to improve the activity of cellulases, which catalyze the hydrolysis of insoluble cellulose, have been hindered by uncertainty surrounding the mechanistic origins of rate-limiting phenomena and by an incomplete understanding of complementary enzyme function. In particular, direct kinetic measurements of individual steps occurring after enzymes adsorb to the cellulose surface have proven to be experimentally elusive. This work describes an experimental and analytical approach, derived from a detailed mechanistic model of cellobiohydrolase action, for determining rates of initial- and processive-cut product generation by Trichoderma longibrachiatum cellobiohydrolase I (TlCel7A) as it catalyzes the hydrolysis of bacterial microcrystalline cellulose (BMCC) alone and in the presence of Talaromyces emersonii endoglucanase II (TemGH5). This analysis revealed that the rate of TlCel7A-catalyzed hydrolysis of crystalline cellulose is limited by the rate of enzyme complexation with glycan chains, which is shown to be equivalent to the rate of initial-cut product generation. This rate is enhanced in the presence of endoglucanase enzymes. The results confirm recent reports about the role of morphological obstacles in enzyme processivity and also provide the first direct evidence that processive length may be increased by the presence of companion enzymes, including small amounts of TemGH5. The findings of this work indicate that efforts to improve cellobiohydrolase activity should focus on enhancing the enzyme's ability to complex with cellulose chains, and the analysis employed provides a new technique for investigating the mechanism by which companion enzymes influence cellobiohydrolase activity.

Topics: Cellulase; Cellulose; Cellulose 1,4-beta-Cellobiosidase; Fungal Proteins; Gene Expression Regulation, Fungal; Oligosaccharides; Protein Processing, Post-Translational; Substrate Specificity; Talaromyces; Trichoderma; Trioses

The hydrolysis of biomass to fermentable sugars using glycosyl hydrolases such as cellulases and hemicellulases is a limiting and costly step in the conversion of biomass to biofuels. Enhancement in hydrolysis efficiency is necessary and requires improvement in both enzymes and processing strategies. Advances in both areas in turn strongly depend on the progress in developing high-throughput assays to rapidly and quantitatively screen a large number of enzymes and processing conditions. For example, the characterization of various cellodextrins and xylooligomers produced during the time course of saccharification is important in the design of suitable reactors, enzyme cocktail compositions, and biomass pretreatment schemes. We have developed a microfluidic-chip-based assay for rapid and precise characterization of glycans and xylans resulting from biomass hydrolysis. The technique enables multiplexed separation of soluble cellodextrins and xylose oligomers in around 1 min (10-fold faster than HPLC). The microfluidic device was used to elucidate the mode of action of Tm_Cel5A, a novel cellulase from hyperthermophile Thermotoga maritima . The results demonstrate that the cellulase is active at 80 °C and effectively hydrolyzes cellodextrins and ionic-liquid-pretreated switchgrass and Avicel to glucose, cellobiose, and cellotriose. The proposed microscale approach is ideal for quantitative large-scale screening of enzyme libraries for biomass hydrolysis, for development of energy feedstocks, and for polysaccharide sequencing.

Topics: Biofuels; Biomass; Cellulase; Electrophoresis, Capillary; Enzyme Assays; Enzyme Stability; Ionic Liquids; Microfluidic Analytical Techniques; N-Glycosyl Hydrolases; Oligosaccharides; Plants; Temperature; Thermotoga maritima; Time Factors; Xylose

The direct conversion of plant cell wall polysaccharides into soluble sugars is one of the most important reactions on earth, and is performed by certain microorganisms such as Clostridium thermocellum (Ct). These organisms produce extracellular multi-subunit complexes (i.e. cellulosomes) comprising a consortium of enzymes, which contain noncatalytic carbohydrate-binding modules (CBM) that increase the activity of the catalytic module. In the present study, we describe a combined approach by X-ray crystallography, NMR and computational chemistry that aimed to gain further insight into the binding mode of different carbohydrates (cellobiose, cellotetraose and cellohexaose) to the binding pocket of the family 11 CBM. The crystal structure of C. thermocellum CBM11 has been resolved to 1.98 A in the apo form. Since the structure with a bound substrate could not be obtained, computational studies with cellobiose, cellotetraose and cellohexaose were carried out to determine the molecular recognition of glucose polymers by CtCBM11. These studies revealed a specificity area at the CtCBM11 binding cleft, which is lined with several aspartate residues. In addition, a cluster of aromatic residues was found to be important for guiding and packing of the polysaccharide. The binding cleft of CtCBM11 interacts more strongly with the central glucose units of cellotetraose and cellohexaose, mainly through interactions with the sugar units at positions 2 and 6. This model of binding is supported by saturation transfer difference NMR experiments and linebroadening NMR studies.

Topics: Bacterial Proteins; Binding Sites; Carbohydrate Conformation; Carbohydrate Sequence; Cellobiose; Cellulase; Cellulose; Clostridium thermocellum; Computer Simulation; Crystallography, X-Ray; Models, Molecular; Molecular Sequence Data; Molecular Structure; Multienzyme Complexes; Nuclear Magnetic Resonance, Biomolecular; Oligosaccharides; Protein Conformation; Substrate Specificity; Tetroses

The structural and thermodynamic basis for carbohydrate-protein recognition is of considerable importance. NCP-1, which is a component of the Piromyces equi cellulase/hemicellulase complex, presents a provocative model for analyzing how structural and mutational changes can influence the ligand specificity of carbohydrate-binding proteins. NCP-1 contains two "family 29" carbohydrate-binding modules designated CBM29-1 and CBM29-2, respectively, that display unusually broad specificity; the proteins interact weakly with xylan, exhibit moderate affinity for cellulose and mannan, and bind tightly to the beta-1,4-linked glucose-mannose heteropolymer glucomannan. The crystal structure of CBM29-2 in complex with cellohexaose and mannohexaose identified key residues involved in ligand recognition. By exploiting this structural information and the broad specificity of CBM29-2, we have used this protein as a template to explore the evolutionary mechanisms that can lead to significant changes in ligand specificity. Here, we report the properties of the E78R mutant of CBM29-2, which displays ligand specificity that is different from that of wild-type CBM29-2; the protein retains significant affinity for cellulose but does not bind to mannan or glucomannan. Significantly, E78R exhibits a stoichiometry of 0.5 when binding to cellohexaose, and both calorimetry and ultracentrifugation show that the mutant protein displays ligand-mediated dimerization in solution. The three-dimensional structure of E78R in complex with cellohexaose reveals the intriguing molecular basis for this "dimeric" binding mode that involves the lamination of the oligosaccharide between two CBM molecules. The 2-fold screw axis of the ligand is mirrored in the orientation of the two protein domains with adjacent sugar rings stacking against the equivalent aromatic residues in the binding site of each protein molecule of the molecular sandwich. The sandwiching of an oligosaccharide chain between two protein modules, leading to ligand-induced formation of the binding site, represents a completely novel mechanism for protein-carbohydrate recognition that may mimic that displayed by naturally dimeric protein-carbohydrate interactions.

Topics: Base Sequence; Binding Sites; Carbohydrate Metabolism; Carbohydrates; Cellulase; Crystallography, X-Ray; Dimerization; DNA, Fungal; Fungal Proteins; Glycoside Hydrolases; Ligands; Macromolecular Substances; Models, Molecular; Multienzyme Complexes; Mutagenesis, Site-Directed; Oligosaccharides; Piromyces; Thermodynamics

Carbohydrate-protein recognition is central to many biological processes. Enzymes that act on polysaccharide substrates frequently contain noncatalytic domains, "carbohydrate-binding modules" (CBMs), that target the enzyme to the appropriate substrate. CBMs that recognize specific plant structural polysaccharides are often able to accommodate both the variable backbone and the side-chain decorations of heterogeneous ligands. "CBM29" modules, derived from a noncatalytic component of the Piromyces equi cellulase/hemicellulase complex, provide an example of this selective yet flexible recognition. They discriminate strongly against some polysaccharides while remaining relatively promiscuous toward both beta-1,4-linked manno- and cello-oligosaccharides. This feature may reflect preferential, but flexible, targeting toward glucomannans in the plant cell wall. The three-dimensional structure of CBM29-2 and its complexes with cello- and mannohexaose reveal a beta-jelly-roll topology, with an extended binding groove on the concave surface. The orientation of the aromatic residues complements the conformation of the target sugar polymer while accommodation of both manno- and gluco-configured oligo- and polysaccharides is conferred by virtue of the plasticity of the direct interactions from their axial and equatorial 2-hydroxyls, respectively. Such flexible ligand recognition targets the anaerobic fungal complex to a range of different components in the plant cell wall and thus plays a pivotal role in the highly efficient degradation of this composite structure by the microbial eukaryote.

Topics: Binding Sites; Carbohydrate Sequence; Carbohydrates; Cellulase; Crystallography, X-Ray; Fungal Proteins; Galactose; Glycoside Hydrolases; Ligands; Mannans; Models, Molecular; Molecular Sequence Data; Oligosaccharides; Piromyces; Protein Structure, Tertiary; Recombinant Fusion Proteins; Substrate Specificity

A new bifunctionalized cellohexaose derivative was synthesized as a specific substrate for continuous assay of cellulases by resonance energy transfer. This cellohexaoside has a naphthalene moiety (EDANS) as a fluorescent energy donor at the reducing end and a 4-(4'-dimethylaminobenzeneazo)-benzene derivative as an acceptor chromophore at the non-reducing end. The key steps for the preparation of the target molecule involved transglycosylation reactions of cellobiosyl and cellotetraosyl fluoride donors onto cellobiosyl acceptors catalysed by the E197A mutant of cellulase Cel7B from Humicola insolens. Upon digestion with various cellulases, the energy transfer was disrupted and an increase of fluorescence was observed.

Topics: Cellulase; Energy Transfer; Fluorometry; Indicators and Reagents; Naphthalenesulfonates; Oligosaccharides; Sensitivity and Specificity; Substrate Specificity

The crystal structure of the Clostridium cellulovorans carbohydrate-binding module (CBM) belonging to family 17 has been solved to 1.7 A resolution by multiple anomalous dispersion methods. CBM17 binds to non-crystalline cellulose and soluble beta-1,4-glucans, with a minimal binding requirement of cellotriose and optimal affinity for cellohexaose. The crystal structure of CBM17 complexed with cellotetraose solved at 2.0 A resolution revealed that binding occurs in a cleft on the surface of the molecule involving two tryptophan residues and several charged amino acids. Thermodynamic binding studies and alanine scanning mutagenesis in combination with the cellotetraose complex structure allowed the mapping of the CBM17 binding cleft. In contrast to the binding groove characteristic of family 4 CBMs, family 17 CBMs appear to have a very shallow binding cleft that may be more accessible to cellulose chains in non-crystalline cellulose than the deeper binding clefts of family 4 CBMs. The structural differences in these two modules may reflect non-overlapping binding niches on cellulose surfaces.

Topics: Alanine; Binding Sites; Calorimetry; Cellulase; Cellulose; Clostridium; Crystallography, X-Ray; Hydrogen Bonding; Ligands; Models, Molecular; Mutation; Oligosaccharides; Protein Binding; Protein Structure, Quaternary; Protein Structure, Secondary; Protein Structure, Tertiary; Static Electricity; Substrate Specificity; Tetroses; Thermodynamics; Titrimetry; Trioses; Tryptophan

Cellulase Cel48F from Clostridium cellulolyticum was described as a processive endo-cellulase. The active site is composed of a 25 A long tunnel which is followed by an open cleft. During the processive action, the cellulose substrate has to slide through the tunnel to continuously supply the leaving group site with sugar residues after the catalytic cleavage. To study this processive action in the tunnel, the native catalytic module of Cel48F and the inactive mutant E55Q, have been cocrystallized with cellobiitol, two thio-oligosaccharide inhibitors (PIPS-IG3 and IG4) and the cello-oligosaccharides cellobiose, -tetraose and -hexaose. Seven sub-sites in the tunnel section of the active center could be identified and three of the four previously reported sub-sites in the open cleft section were reconfirmed. The sub-sites observed for the thio-oligosaccharide inhibitors and oligosaccharides, respectively, were located at two different positions in the tunnel corresponding to a shift in the chain direction of about a half sugar subunit. These two positions have different patterns of stacking interactions with aromatic residues present in the tunnel. Multiple patterns are not observed in nonprocessive endo-cellulases, where only one sugar position is favored by aromatic stacking. It is therefore proposed that the aromatic residues serve as lubricating agents to reduce the sliding barrier in the processive action.

Topics: Binding Sites; Cellobiose; Cellulase; Cellulose; Clostridium; Crystallography, X-Ray; Enzyme Inhibitors; Hydrogen Bonding; Hydrolysis; Macromolecular Substances; Models, Chemical; Models, Molecular; Mutagenesis, Site-Directed; Oligosaccharides; Substrate Specificity; Tetroses

The C-terminal carbohydrate-binding module (CBM17) from Clostridium cellulovorans cellulase 5A is a beta-1,4-glucan binding module with a preference for soluble chains. CBM17 binds to phosphoric acid swollen Avicel (PASA) and Avicel with association constants of 2.9 (+/-0.2) x 10(5) and 1.6 (+/-0.2) x 10(5) M(-1), respectively. The capacity values for PASA and Avicel were 11.9 and 0.4 micromol/g of cellulose, respectively. CBM17 did not bind to crystalline cellulose. CBM17 bound tightly to soluble barley beta-glucan and the derivatized celluloses HEC, EHEC, and CMC. The association constants for binding to barley beta-glucan, HEC, and EHEC were approximately 2.0 x 10(5) M(-1). Significant binding affinities were found for cello-oligosaccharides greater than three glucose units in length. The affinities for cellotriose, cellotetraose, cellopentaose, and cellohexaose were 1.2 (+/-0.3) x 10(3), 4.3 (+/-0.4) x 10(3), 3.8 (+/-0.1) x 10(4), and 1.5 (+/-0.0) x 10(5) M(-1), respectively. Fluorescence quenching studies and N-bromosuccinimide modification indicate the participation of tryptophan residues in ligand binding. The possible architecture of the ligand-binding site is discussed in terms of its binding specificity, affinity, and the participation of tryptophan residues.

Topics: Amino Acid Motifs; Amino Acid Sequence; Amino Acids; Bacterial Proteins; Binding Sites; Carrier Proteins; Cellulase; Clostridium; Glucans; Hydrogen-Ion Concentration; Molecular Sequence Data; Oligosaccharides; Peptide Fragments; Polysaccharides; Sequence Homology, Amino Acid; Sodium Chloride; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Substrate Specificity

To investigate the mode of action of cellulose-binding domains (CBDs), the Type II CBD from Pseudomonas fluorescens subsp. cellulosa xylanase A (XYLACBD) and cellulase E (CELECBD) were expressed as individual entities or fused to the catalytic domain of a Clostridium thermocellum endoglucanase (EGE). The two CBDs exhibited similar Ka values for bacterial microcrystalline cellulose (CELECBD, 1.62x10(6) M-1; XYLACBD, 1.83x10(6) M-1) and acid-swollen cellulose (CELECBD, 1.66x10(6) M-1; XYLACBD, 1.73x10(6) M-1). NMR spectra of XYLACBD titrated with cello-oligosaccharides showed that the environment of three tryptophan residues was affected when the CBD bound cellohexaose, cellopentaose or cellotetraose. The Ka values of the XYLACBD for C6, C5 and C4 cello-oligosaccharides were estimated to be 3.3x10(2), 1.4x10(2) and 4.0x10(1) M-1 respectively, suggesting that the CBD can accommodate at least six glucose molecules and has a much higher affinity for insoluble cellulose than soluble oligosaccharides. Fusion of either the CELECBD or XYLACBD to the catalytic domain of EGE potentiated the activity of the enzyme against insoluble forms of cellulose but not against carboxymethylcellulose. The increase in cellulase activity was not observed when the CBDs were incubated with the catalytic domain of either EGE or XYLA, with insoluble cellulose and a cellulose/hemicellulose complex respectively as the substrates. Pseudomonas CBDs did not induce the extension of isolated plant cell walls nor weaken cellulose paper strips in the same way as a class of plant cell wall proteins called expansins. The XYLACBD and CELECBD did not release small particles from the surface of cotton. The significance of these results in relation to the mode of action of Type II CBDs is discussed.

Topics: Bacterial Proteins; Binding Sites; Cellulase; Cellulose; Clostridium; Endo-1,4-beta Xylanases; Kinetics; Magnetic Resonance Spectroscopy; Oligosaccharides; Pseudomonas fluorescens; Recombinant Fusion Proteins; Xylosidases

The solution structure of a synthetic 38-residue cellulose-binding domain (CBD) of endoglucanase I from Trichoderma reesei (CBD(EGI)) was determined by two-dimensional 1H-NMR spectroscopy. 100 structures were generated from a total of 599 NOE derived distance restraints and 28 phi and 14 chi dihedral angle restraints. For the final set of 19 selected structures, the rms deviation about the mean structure was 0.83+/-0.26 A for all atoms and 0.50+/-0.22 A for the backbone atoms. The structure of CBD(EGI) was very similar to that of CBD of cellobiohydrolase I from T reesei (CBD(CBHI)). The backbone trace of CBD(EGI) followed closely the irregular triple-stranded antiparallel beta-sheet structure of CBD(CBHI). Moreover, apart from the different side chains of Trp7 (CBD(EGI)) and Tyr5 (CBD(CBHI)), the cellulose-binding face of CBD(EGI) was similar to that of CBD(CBHI) within the precision of the structures. Finally, the interaction between CBD(EGI) and soluble sugars was investigated using cellopentaose and cellohexaose as substrates. Experiments showed that the interactions between CBD(EGI) and cellobiose units of sugars are specific, supporting the previously presented model for the CBD binding to crystalline cellulose.

Topics: Amino Acid Sequence; Binding Sites; Carbohydrate Sequence; Cellulase; Cellulose; Cellulose 1,4-beta-Cellobiosidase; Fungal Proteins; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Oligosaccharides; Protein Binding; Protein Structure, Secondary; Sequence Alignment; Substrate Specificity; Trichoderma

Most Trichoderma reesei cellulases consist of a catalytic and a cellulose binding domain (CBD) joined by a linker. We have used cellohexaose as a model compound for the glucose chain to investigate the interaction between the soluble enzyme and cellulose. The binding of cellohexaose to family I CBDs was studied by NMR spectroscopy. CBDs cause line broadening effects and decreasing T2 relaxation times for certain cellohexaose resonances, whereas there are no effects in the presence of a mutant which binds weakly to cellulose. Yet it remains uncertain how well the soluble cellooligosaccharide mimics the binding of CBD to the cellulose.

Topics: Binding Sites; Carbohydrate Sequence; Cellulase; Cellulose; Cloning, Molecular; Escherichia coli; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Molecular Structure; Oligosaccharides; Peptide Fragments; Point Mutation; Protein Conformation; Protein Engineering; Recombinant Proteins; Trichoderma

The structures of the Glu140-->Gln mutant of the Clostridium thermocellum endoglucanase CelC in unliganded form (CelC(E140Q)) and in complex with cellohexaose (CelC(E140Q)-Gl(C6)) have been refined to crystallographic R-factors of 19.4% at 1.9 A and 17.8% at 2.3 A resolution, respectively. The structure of CelC(E140Q)-Gl(C6) complex shows two D-glucosyl residues bound to the non-reducing end of the substrate-binding cleft. Comparison of the unliganded and complexes structures reveals conformational changes due to substrate binding, including a significant reorientation of the loop 138-141 which carries the general acid/base catalyst Glu140 in wild-type CelC. Endoglucanase CelC, a family 5 glycohydrolase, exhibits a (beta/alpha)8-fold with an additional subdomain of 54 amino acids inserted between beta-strand 6 and alpha-helix 6. Seven amino acid residues (Arg46, His90, Asn139, Glu140, His198, Tyr200, and Glu280) located close to the catalytic reaction center are strictly conserved in family 5 cellulases. Only three of these residues (His90, Gln140 and Glu280) make direct contacts with the substrate, but all participate in a network of hydrogen bonds which contribute to the stability of the active site architecture and may influence the protonation state of the two catalytic residues. Residue Trp313, which interacts with the nucleophile Glu280 and is within hydrogen bonding distance of the substrate, is involved in a non-proline cis-peptide bond. An aromatic residue occurs at an equivalent position in many other (beta/alpha)8-barrel glycosidases; the presence of a cis-peptide bond at this position in the structures of family 1 beta-glucosidases, family 2 beta-galactosidases, family 5 cellulases, family 17 beta-glucanases, and family 18 chitinases provides further evidence of an evolutionary relationship between glycosyl hydrolases with a (beta/alpha)8- architecture.

Topics: Binding Sites; Cellulase; Clostridium; Computer Graphics; Crystallography, X-Ray; Evolution, Molecular; Glycoside Hydrolases; Hydrogen Bonding; Models, Molecular; Mutation; Oligosaccharides; Protein Binding; Protein Conformation; Protein Structure, Secondary

The hydrolysis of soluble cello-oligosaccharides, with a degree of polymerisation of 4-6, catalysed by cellobiohydrolase II from Trichoderma reesei was studied using 1H-NMR spectroscopy and HPLC. The experimental progress curves were analysed by fitting numerically integrated kinetic equations, which provided cleavage patterns and kinetic constants for each oligosaccharide. This analysis procedure accounts for product inhibition and avoids the initial slope approximation. No glucose was detected at the beginning of the reaction indicating that only the internal glycosidic linkages are attacked. For cellotetraose only the second glycosidic linkage was cleaved. For cellopentaose and cellohexaose the second and the third glycosidic linkage from the non-reducing end were cleaved with approximately equal probability. The degradation rates of these cello-oligosaccharides, 1-12 s-1 at 27 degrees C, are about 10-100 times faster than for the 4-methylumbelliferyl substituted analogs or for collotriose. No intermediate products larger than cellotriose were released. The degradation rate for cellotetraose were higher than its off-rate, which accounts for the processive degradation of cellohexaose. A high cellohexaose/enzyme ratio caused slow reversible inactivation of the enzyme.

Topics: Binding Sites; Cellulase; Cellulose; Cellulose 1,4-beta-Cellobiosidase; Chromatography, High Pressure Liquid; Hydrolysis; Kinetics; Magnetic Resonance Spectroscopy; Oligosaccharides; Substrate Specificity; Tetroses; Trichoderma