Domain coupling in a multimodular cellobiohydrolase CbhA from Clostridium thermocellum

Cellobiohydrolase A (CbhA) from Clostridium thermocellum is composed of an N-terminal carbohydrate-binding domain 4 (CBD4), an immunoglobulin-like domain (Ig), a glycoside hydrolase 9 (GH9), X1(1) and X1(2) domains, a CBD3, and a dockerin domain. All domains, except the Ig, bind Ca2+. The following constructs were made: X1(2), X1(1)X1(2), CBD3, X1(1)X1(2)-CBD3, Ig, GH9, Ig-GH9, Ig-GH9-X1(1)X1(2), and Ig-GH9-X1(1)X1(2)-CBD3. Interactions between domains in (1) buffer, (2) with Ca2+, or (3) ethylenediaminetetraacetic acid (EDTA) were studied by differential scanning calorimetry. Thermal unfoldings of all constructs were irreversible. Calcium increased T(d) and cooperativity of unfolding. Multi-domain constructs exhibited more cooperative unfolding in buffer and in the presence of EDTA than did individual domains. They denatured by mechanism simpler than expected from their modular architecture. The results indicate that domain coupling in thermophilic proteins constitutes a significant stabilizing factor.     

Multidomain Structure and Cellulosomal Localization of the Clostridium thermocellum Cellobiohydrolase CbhA

The nucleotide sequence of the Clostridium thermocellum F7 cbhA gene, coding for the cellobiohydrolase CbhA, has been determined. An open reading frame encoding a protein of 1,230 amino acids was identified. Removal of a putative signal peptide yields a mature protein of 1,203 amino acids with a molecular weight of 135,139. Sequence analysis of CbhA reveals a multidomain structure of unusual complexity consisting of an N-terminal cellulose binding domain (CBD) homologous to CBD family IV, an immunoglobulin-like β-barrel domain, a catalytic domain homologous to cellulase family E1, a duplicated domain similar to fibronectin type III (Fn3) modules, a CBD homologous to family III, a highly acidic linker region, and a C-terminal dockerin domain. The cellulosomal localization of CbhA was confirmed by Western blot analysis employing polyclonal antibodies raised against a truncated enzymatically active version of CbhA. CbhA was identified as cellulosomal subunit S3 by partial amino acid sequence analysis. Comparison of the multidomain structures indicates striking similarities between CbhA and a group of cellulases from actinomycetes. Average linkage cluster analysis suggests a coevolution of the N-terminal CBD and the catalytic domain and its spread by horizontal gene transfer among gram-positive cellulolytic bacteria.     

Isolation and properties of a major cellobiohydrolase from the cellulosome of Clostridium thermocellum.

In the anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum, efficient solubilization of the insoluble cellulose substrate is accomplished largely through the action of a cellulose-binding multienzyme complex, the cellulosome. A major cellobiohydrolase activity from the cellulosome has been traced to its Mr 75,000 S8 subunit, and an active fragment of this subunit was prepared by a novel procedure involving limited proteolytic cleavage. The truncated Mr 68,000 fragment, termed S8-tr, was purified by gel filtration and high-performance ion-exchange chromatography. The purified protein adsorbed weakly to amorphous cellulose, and its enzymatic action yielded cellobiose as the major end product from both amorphous and crystalline cellulose preparations. The high ratio of exo- to endo-beta-glucanase activities was supported by viscosimetric measurements. The use of model substrates showed that the smallest cellodextrin to be degraded was cellotetraose, but cellopentaose was degraded at a much greater rate. Cellobiose dramatically inhibited the cellulolytic activities. In the absence of calcium or other bivalent metal ions, both the truncated cellobiohydrolase activity of S8-tr and the true cellulase activity of the parent cellulosome were relatively unstable at temperatures above 50 degrees C. Cysteine further enhanced the stabilizing effect of calcium. This is the first report of a defined cellobiohydrolase in C. thermocellum. Its association with the cellulosome and the correspondence of several of their major distinctive properties suggest that this cellobiohydrolase plays a key role in the solubilization of cellulose by the intact cellulosomal complex.     

Structural basis for the substrate specificity of the feruloyl esterase domain of the cellulosomal xylanase Z from Clostridium thermocellum

Feruloyl esterases function in the cleavage of ferulic acid's bonds to arabinoxylan and pectin where the ferulic acid moieties cross-link the layers of polysaccharide chains within hemicellulose. This work presents the crystal structure of FAE_XynZ, the domain of Clostridium thermocellum's cellulosomal xylanase Z that displays feruloyl esterase activity. The structure was obtained via multiple isomorphous replacement with anomalous scattering (MIRAS) using three heavy atom derivatives and refined against X-ray diffraction data of up to 1.75 A resolution. The R-value of the final model was 0.187 (R(free) = 0.21). FAE_XynZ displays an eight-stranded alpha/beta-fold with the characteristic "catalytic triad" at the heart of the active site. To define the substrate specificity determinants of the enzyme, the crystal structures of FAE_XynZ and the inactive FAE_XynZ(S172A) mutant were determined in complexes with the feruloyl-arabinoxylans FAXX and FAX(3), respectively. In the complex crystals, the ferulic acid moieties are clearly recognizable and allowed identification of the hydrophobic binding pocket. The carbohydrate part of both substrates is not visible in either structure. The location of the putative carbohydrate binding-pocket was inferred based on the location and orientation of the adjacent ferulic acid molecule. Five of the six residues lining the pocket were found to be conserved in FAE A from Orpinomyces sp., which further supports the proposed role of these amino acids.     

The Fibronectin Type 3-Like Repeat from the Clostridium thermocellum Cellobiohydrolase CbhA Promotes Hydrolysis of Cellulose by Modifying Its Surface

Fibronectin type 3 homology domains (Fn3) as found in the cellobiohydrolase CbhA of Clostridium thermocellum are common among bacterial extracellular glycohydrolases. The function of these domains is not clear. CbhA is modular and composed of an N-terminal family IV carbohydrate-binding domain (CBDIV), an immunoglobulin-like domain, a family 9 glycosyl hydrolase catalytic domain (Gh9), two Fn3-like domains (Fn3_1,2), a family III carbohydrate-binding domain (CBDIII), and a dockerin domain. Efficiency of cellulose hydrolysis by truncated forms of CbhA increased in the following order: Gh9 (lowest efficiency), Gh9-Fn3_1,2 (more efficient), and Gh9-Fn3_1,2-CBDIII (greatest efficiency). Thermostability of the above constructs decreased in the following order: Gh9 (most stable), Gh9-Fn3_1,2, and then Gh9-Fn3_1,2-CBDIII (least stable). Mixing of Orpinomyces endoglucanase CelE with Fn3_1,2, or Fn3_1,2-CBDIII increased efficiency of hydrolysis of acid-swollen cellulose (ASC) and filter paper. Scanning electron microscopic studies of filter paper treated with Fn3_1,2, Fn3_1,2-CBDIII, or CBDIII showed that the surface of the cellulose fibers had been loosened up and crenellated by Fn3_1,2 and Fn3_1,2-CBDIII and to a lesser extent by CBDIII. X-ray diffraction analysis did not reveal changes in the crystallinity of the filter paper. CBDIII bound to ASC and filter paper with capacities of 2.45 and 0.73 μmoles g⁻¹ and relative affinities (K_r) of 1.12 and 2.13 liters g⁻¹, respectively. Fn3_1,2 bound weakly to both celluloses. Fn3_1,2-CBD bound to ASC and filter paper with capacities of 3.22 and 0.81 μmoles g⁻¹ and K_rs of 1.14 and 1.98 liters g⁻¹, respectively. Fn3_1,2 and CBDIII contained 2 and 1 mol of calcium per mol, respectively. The results suggest that Fn3_1,2 aids the hydrolysis of cellulose by modifying its surface. This effect is enhanced by the presence of CBDIII, which increases the concentration of Fn3_1,2 on the cellulose surface.     

Subcellulosome preparation with high cellulase activity from Clostridium thermocellum

We have prepared a much simpler cellulase preparation than that of cellulosomes from the extracellular broth of Clostridium thermocellum. This "subcellulosome" preparation from C. thermocellum was obtained by column chromatography on CM-Bio-Gel A and then on a lectin-affinity material (Jacalin). The subcellulosome preparation is a macromolecular complex, composed of six main protein subunits (molecular weight, 210,000 to 58,000) revealed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activities of carboxymethylcellulase (CMCase) and Avicelase are 15- and 8-fold-higher, respectively, than those of crude extracellular cellulase. We could not further fractionate this preparation without denaturing it. The optimum pH and temperature of the subcellulosome preparation are 5.5 to 7.0 and 70 degrees C for CMCase and 5.5 to 7.0 and 65 degrees C for Avicelase. The subcellulosome preparation acted on various types of carboxymethyl cellulose, cellulose, and p-nitrophenyl-beta-D-cellobioside but not on p-nitrophenyl-beta-D-glucoside. Sulfhydryl reagents and N-bromosuccinimide inhibited both CMCase and Avicelase activities, whereas EDTA and o-phenanthroline inhibited Avicelase activity only.     

Restriction endonuclease activity in Clostridium thermocellum and Clostridium thermosaccharolyticum

 Clostridium thermocellum cell extracts exhibit specific endonuclease activity with very little non-specific exonuclease activity at 55°C. The Dam methylation system of Escherichia coli offers complete protection from digestion by C. thermocellum ATCC 27405 cell extracts for all DNA tested (totaling >100 kb, insuring that most potential restriction sequences have been exposed). Based on both the Dam recognition sequence and the similarity of cell extract and MboI DNA digests, the C. thermocellum restriction enzyme recognition sequence appears to be 5′ GATC 3′. Cell extracts made from a second thermophile, C. thermosaccharolyticum ATCC 31960 do not exhibit specific endonuclease activity under the conditions tested. Genomic DNA from C. thermocellum exhibits a Dam⁺ phenotype while genomic DNA from C. thermosaccharolyticum exhibits a Dam^- phenotype. Received: 10 March 1995/Received revision: 4 September 1995/Accepted: 13 September 1995     

Subcellulosome preparation with high cellulase activity from Clostridium thermocellum.

We have prepared a much simpler cellulase preparation than that of cellulosomes from the extracellular broth of Clostridium thermocellum. This "subcellulosome" preparation from C. thermocellum was obtained by column chromatography on CM-Bio-Gel A and then on a lectin-affinity material (Jacalin). The subcellulosome preparation is a macromolecular complex, composed of six main protein subunits (molecular weight, 210,000 to 58,000) revealed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activities of carboxymethylcellulase (CMCase) and Avicelase are 15- and 8-fold-higher, respectively, than those of crude extracellular cellulase. We could not further fractionate this preparation without denaturing it. The optimum pH and temperature of the subcellulosome preparation are 5.5 to 7.0 and 70 degrees C for CMCase and 5.5 to 7.0 and 65 degrees C for Avicelase. The subcellulosome preparation acted on various types of carboxymethyl cellulose, cellulose, and p-nitrophenyl-beta-D-cellobioside but not on p-nitrophenyl-beta-D-glucoside. Sulfhydryl reagents and N-bromosuccinimide inhibited both CMCase and Avicelase activities, whereas EDTA and o-phenanthroline inhibited Avicelase activity only.     

Two proteins with diaphorase activity from Clostridium thermocellum and Moorella thermoacetica

Two high-molecular-weight rubredoxin genes (hrb) from Clostridium thermocellum and Moorella thermoacetica were expressed in Escherichia coli and their translated products (Hrb) were characterized. M. thermoacetica Hrb showed strong diaphorase activity. In contrast, C. thermocellum Hrb, containing neither FMN nor FAD in the molecule, showed flavin reductase activity with moderate diaphorase activity. Both enzymes were optimally active at about 50 degrees C.     

梭热杆菌纤维小体研究进展

梭热杆菌(Clostridium thermocellum)是一种嗜热厌氧细菌,通过分泌大量纤维素酶高效降解纤维素.根据作用纤维素的不同部位,梭热杆菌分泌的纤维素酶分为内切纤维素酶和外切纤维素酶.纤维小体是由支架蛋白、锚定元件、黏合蛋白、纤维素结合域和催化单位组成的复合体,其独特的结构,使得它可以比真菌纤维素酶更紧密地结合到纤维素表面,这个复合结构结合着多种催化单位,而此特殊的结构是梭热杆菌高效降解纤维素的必要条件.近年来,为更深入透彻地了解纤维小体的结构与功能进行了大量的研究工作,现对相关研究进展进行综述,并给出了未来可能的发展方向.     

Structural basis for ligand binding and processivity in cellobiohydrolase Cel6A from Humicola insolens

The enzymatic digestion of cellulose entails intimate involvement of cellobiohydrolases, whose characteristic active-center tunnel contributes to a processive degradation of the polysaccharide. The cellobiohydrolase Cel6A displays an active site within a tunnel formed by two extended loops, which are known to open and close in response to ligand binding. Here we present five structures of wild-type and mutant forms of Cel6A from Humicola insolens in complex with nonhydrolyzable thio-oligosaccharides, at resolutions from 1.7-1.1 A, dissecting the structural accommodation of a processing substrate chain through the active center during hydrolysis. Movement of ligand is facilitated by extensive solvent-mediated interactions and through flexibility in the hydrophobic surfaces provided by a sheath of tryptophan residues.     

The crystal structure and catalytic mechanism of cellobiohydrolase CelS,the major enzymatic component of the Clostridium thermocellum Cellulosome

Cellobiohydrolase CelS plays an important role in the cellulosome, an active cellulase system produced by the thermophilic anaerobe Clostridium thermocellum. The structures of the catalytic domain of CelS in complex with substrate (cellohexaose) and product (cellobiose) were determined at 2.5 and 2.4 A resolution, respectively. The protein folds into an (alpha/alpha)(6) barrel with a tunnel-shaped substrate-binding region. The conformation of the loops defining the tunnel is intrinsically stable in the absence of substrate, suggesting a model to account for the processive mode of action of family 48 cellobiohydrolases. Structural comparisons with other (alpha/alpha)(6) barrel glycosidases indicate that CelS and endoglucanase CelA, a sequence-unrelated family 8 glycosidase with a groove-shaped substrate-binding region, use the same catalytic machinery to hydrolyze the glycosidic linkage, despite a low sequence similarity and a different endo/exo mode of action. A remarkable feature of the mechanism is the absence, from CelS, of a carboxylic group acting as the base catalyst. The nearly identical arrangement of substrate and functionally important residues in the two active sites strongly suggests an evolutionary relationship between the cellobiohydrolase and endoglucanase families, which can therefore be classified into a new clan of glycoside hydrolases.     

Biochemical Characterization and Structural Analysis of a Bifunctional Cellulase/Xylanase from Clostridium thermocellum

We expressed an active form of CtCel5E (a bifunctional cellulase/xylanase from Clostridium thermocellum), performed biochemical characterization, and determined its apo- and ligand-bound crystal structures. From the structures, Asn-93, His-168, His-169, Asn-208, Trp-347, and Asn-349 were shown to provide hydrogen-bonding/hydrophobic interactions with both ligands. Compared with the structures of TmCel5A, a bifunctional cellulase/mannanase homolog from Thermotoga maritima, a flexible loop region in CtCel5E is the key for discriminating substrates. Moreover, site-directed mutagenesis data confirmed that His-168 is essential for xylanase activity, and His-169 is more important for xylanase activity, whereas Asn-93, Asn-208, Tyr-270, Trp-347, and Asn-349 are critical for both activities. In contrast, F267A improves enzyme activities.     

Electrotransformation of Clostridium thermocellum

Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 ± 0.5) × 10⁵ transformants per μg of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 ± 1.8) × 10⁴ transformants per μg of plasmid DNA for strain ATCC 27405 and ~1 × 10³ transformants per μg of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was ~50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in C. thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.     

Electrotransformation of Clostridium thermocellum

Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 +/- 0.5) x 10(5) transformants per micro g of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 +/- 1.8) x 10(4) transformants per micro g of plasmid DNA for strain ATCC 27405 and approximately 1 x 10(3) transformants per micro g of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was approximately 50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in C. thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.     

Structural basis for the function of Clostridium difficile toxin B

Toxin B is a member of the family of large clostridial cytotoxins which are of great medical importance. Its catalytic fragment was crystallized in the presence of UDP-glucose and Mn2+. The structure was determined at 2.2 A resolution, showing that toxin B belongs to the glycosyltransferase type A family. However, toxin B contains as many as 309 residues in addition to the common chainfold, which most likely contribute to the target specificity. A superposition with other glycosyltransferases shows the expected positions of the acceptor oxygen atom during glucosyl transfer and indicates further that the reaction proceeds probably along a single-displacement pathway. The C1' donor carbon atom position is defined by the bound UDP and glucose. It assigns the surface area of toxin B that forms the interface to the target protein during the modifying reaction. A docking attempt brought the known acceptor atom, Thr37 O(gamma1) of the switch I region of the RhoA:GDP target structure, near the expected position. The relative orientation of the two proteins was consistent with both being attached to a membrane. Sequence comparisons between toxin B variants revealed that the highest exchange rate occurs around the active center at the putative docking interface, presumably due to a continuous hit-and-evasion struggle between Clostridia and their eukaryotic hosts.     

Structural basis for thermostability in aporubredoxins from Pyrococcus furiosus and Clostridium pasteurianum

The structures of apo- and holorubredoxins from Pyrococcus furiosus (PfRd) and Clostridium pasteurianum (CpRd) have been investigated and compared using residual dipolar couplings to probe the origin of thermostability. In the native, metal (Fe or Zn) containing form, both proteins can maintain native structure at very high temperatures (>70 degrees C) for extended periods of time. Significant changes in either structure or backbone dynamics between 25 and 70 degrees C are not apparent for either protein. A kinetic difference with respect to metal loss is observed as in previous studies, but the extreme stability of both proteins in the presence of metal makes thermodynamic differences difficult to monitor. In the absence of metal, however, a largely reversible thermal denaturation can be monitored, and a comparison of the two apoproteins can offer insights into the origin of stability. Below denaturation temperatures apo-PfRd is found to have a structure nearly identical to that of the native holo form, except immediately adjacent to the metal binding site. In contrast, apo-CpRd is found to have a structure distinctly different from that of its holo form at low temperatures. This structure is rapidly lost upon heating, unfolding at approximately 40 degrees C. A PfRd mutant with the hydrophobic core mutated to match that of CpRd shows no change in thermostability in the metal-free state. A metal-free chimera with residues 1-15 of CpRd and the remaining 38 residues of PfRd is severely destabilized and is unfolded at 25 degrees C. Hence, the hydrophobic core does not seem to be the key determinant of thermostability; instead, data point to the hydrogen bond network centered on the first 15 residues or the interaction of these 15 residues with other parts of the protein as a possible contributor to the thermostability.     

Purification and properties of cellodextrin phosphorylase from Clostridium thermocellum

Cellodextrin phosphorylase [EC 2.4.1.49] was purified 129-fold, with a yield of 22.9%, to electrophoretic and column chromatographic homogeneity from a cell extract of Clostridium thermocellum ATCC 27405 by a procedure which included streptomycin treatment, ammonium sulfate precipitation, DEAE-Toyopearl 650 M, and Toyopearl HW-55F column chromatography. The molecular weight of the enzyme was estimated to be 200,000 by gel filtration and 105,000 by SDS-PAGE, suggesting that it consisted of two identical subunits. It was suggested by spectrophotometric and chemical analysis that the enzyme contained no pyridoxal 5′-phosphate. The enzyme was inactivated by N-ethylmaleimide and activated by dithiothreitol, indicating that the exposed thiol group(s) was important for the enzymatic activity. The enzyme could synthesize at least cellotriose, cellotetraose, and cellopentaose as detectable cellodextrins, showing that it might possibly be a good tool for the synthesis of cellodextrins.