Processivity, Substrate Binding, and Mechanism of Cellulose Hydrolysis by Thermobifida fusca Cel9A

Thermobifida fusca Cel9A-90 is a processive endoglucanase consisting of a family 9 catalytic domain (CD), a family 3c cellulose binding module (CBM3c), a fibronectin III-like domain, and a family 2 CBM. This enzyme has the highest activity of any individual T. fusca enzyme on crystalline substrates, particularly bacterial cellulose (BC). Mutations were introduced into the CD or the CBM3c of Cel9A-68 using site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli; purified; and tested for activity on four substrates, ligand binding, and processivity. The results show that H125 and Y206 play an important role in activity by forming a hydrogen bonding network with the catalytic base, D58; another important supporting residue, D55; and Glc(−1) O1. R378, a residue interacting with Glc(+1), plays an important role in processivity. Several enzymes with mutations in the subsites Glc(−2) to Glc(−4) had less than 15% activity on BC and markedly reduced processivity. Mutant enzymes with severalfold-higher activity on carboxymethyl cellulose (CMC) were found in the subsites from Glc(−2) to Glc(−4). The CBM3c mutant enzymes, Y520A, R557A/E559A, and R563A, had decreased activity on BC but had wild-type or improved processivity. Mutation of D513, a conserved residue at the end of the CBM, increased activity on crystalline cellulose. Previous work showed that deletion of the CBM3c abolished crystalline activity and processivity. This study shows that it is residues in the catalytic cleft that control processivity while the CBM3c is important for loose binding of the enzyme to the crystalline cellulose substrate.     

Processivity, substrate binding, and mechanism of cellulose hydrolysis by Thermobifida fusca Cel9A

Thermobifida fusca Cel9A-90 is a processive endoglucanase consisting of a family 9 catalytic domain (CD), a family 3c cellulose binding module (CBM3c), a fibronectin III-like domain, and a family 2 CBM. This enzyme has the highest activity of any individual T. fusca enzyme on crystalline substrates, particularly bacterial cellulose (BC). Mutations were introduced into the CD or the CBM3c of Cel9A-68 using site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli; purified; and tested for activity on four substrates, ligand binding, and processivity. The results show that H125 and Y206 play an important role in activity by forming a hydrogen bonding network with the catalytic base, D58; another important supporting residue, D55; and Glc(-1) O1. R378, a residue interacting with Glc(+1), plays an important role in processivity. Several enzymes with mutations in the subsites Glc(-2) to Glc(-4) had less than 15% activity on BC and markedly reduced processivity. Mutant enzymes with severalfold-higher activity on carboxymethyl cellulose (CMC) were found in the subsites from Glc(-2) to Glc(-4). The CBM3c mutant enzymes, Y520A, R557A/E559A, and R563A, had decreased activity on BC but had wild-type or improved processivity. Mutation of D513, a conserved residue at the end of the CBM, increased activity on crystalline cellulose. Previous work showed that deletion of the CBM3c abolished crystalline activity and processivity. This study shows that it is residues in the catalytic cleft that control processivity while the CBM3c is important for loose binding of the enzyme to the crystalline cellulose substrate.     

Site-directed mutation of noncatalytic residues of Thermobifida fusca exocellulase Cel6B.

Fifteen mutant genes in six loop residues and eight mutant genes in five conserved noncatalytic active site residues of Thermobifida fusca Cel6B were constructed, cloned and expressed in Escherichia coli or Streptomyces lividans. The mutant enzymes were assayed for catalytic activity on carboxymethyl cellulose (CMC), swollen cellulose (SC), filter paper (FP), and bacterial microcrystalline cellulose (BMCC) as well as cellotetraose, cellopentaose, and 2, 4-dinitrophenyl-beta-D-cellobioside. They were also assayed for ligand binding, enzyme processivity, thermostability, and cellobiose feedback inhibition. Two double Cys mutations that formed disulfide bonds across two tunnel forming loops were found to significantly weaken binding to ligands, lower all activities, and processivity, demonstrating that the movement of these loops is important but not essential for Cel6B function. Two single mutant enzymes, G234S and G284P, had higher activity on SC and FP, and the double mutant enzyme had threefold and twofold higher activity on these substrates, respectively. However, synergism with endocellulase T. fusca Cel5A was not increased with these mutant enzymes. All mutant enzymes with lower activity on filter paper, BMCC, and SC had lower processivity. This trend was not true for CMC, suggesting that processivity in Cel6B is a key factor in the hydrolysis of insoluble and crystalline cellulose. Three mutations (E495D, H326A and W329C) located near putative glycosyl substrate subsites -2, +1 and +2, were found to significantly increase resistance to cellobiose feedback inhibition. Both the A229V and L230C mutations specifically decreased activity on BMCC, suggesting that BMCC hydrolysis has a different rate limiting step than the other substrates. Most of the mutant enzymes had reduced thermostability although Cel6B G234S maintained wild-type thermostability. The properties of the different mutant enzymes provide insight into the catalytic mechanism of Cel6B.     

Cooperative and competitive binding in synergistic mixtures of Thermobifida fusca cellulases Cel5A,Cel6B,and Cel9A

Synergism between cellulases facilitates efficient hydrolysis of microcrystalline cellulose. We hypothesize that the effects of synergism, observed as enhanced extents of hydrolysis, are related to cellulase binding to the substrate in mixtures. In this study, direct measurements of bound concentrations of fluorescence-labeled T. fusca Cel5A, Cel6B, and Cel9A on bacterial microcrystalline cellulose were used to study binding behaviors of cellulases in binary component reactions. The accuracy of the determination of fluorescence-labeled cellulase concentrations in binary component mixtures was in the range of 7-9%. Data at 5 degrees C show that binding levels of cellulases in mixture reactions are only 22-70% of the binding levels in single component reactions. At 50 degrees C, however, most of the cellulase components in the same mixtures bound to extents of 40-126% higher than in the corresponding single component reactions. The degrees of synergistic effect (DSE) observed for the reactions at 50 degrees C were greater than 1, indicating that the components in the mixture acted synergistically, whereas DSE < 1 was generally observed for the reactions at 5 degrees C indicating anti-synergistic behavior. Degrees of synergistic binding (DSB) were also calculated, where anti-synergistic mixtures had DSB < 1 and synergistic mixtures had DSB>1. We conclude that the lower extents of binding at 5 degrees C are due to competition for binding sites by the cellulase components in the mixtures and the enhanced binding extents at 50 degrees C are due to increased availability of binding sites on the substrates brought about by the higher extents of hydrolysis.     

Binding mechanisms for Thermobifida fusca Cel5A,Cel6B,and Cel48A cellulose-binding modules on bacterial microcrystalline cellulose

The family II cellulose-binding modules (CBM) from Thermobifida fusca Cel5A and Cel48A were cloned in the Escherichia coli/Streptomyces shuttle vector pD730, and the plasmids were transformed into Streptomyces lividans TKM31. CBM(Cel5A), and CBM(Cel48A), CBM(Cel6B) were expressed and purified from S. lividans. The molecular masses were determined by mass spectrometry, and the values were 10595 +/- 2, 10915 +/- 2, and 11291 +/- 2 Da for CBM(Cel5A), CBM(Cel6B), and CBM(Cel48A), respectively. Three different binding models (Langmuir, Interstice Penetration, and Interstice Saturation) were tested to describe the binding isotherms of these CBMs on bacterial microcrystalline cellulose (BMCC). The experimental binding isotherms of T. fusca family II CBMs on BMCC are best modeled by the Interstice Saturation model, which includes binding to the constrained interstice surface of BMCC as well as traditional Langmuir binding on the freely accessible surface. The Interstice Saturation model consists of three different steps (Langmuir binding, interstice binding, and interstice saturation). Full reversibility only occurred in the Langmuir region. The irreversibility in the interstice binding and saturation regions probably was caused by interstice entrapment. Temperature shift experiments in different binding regions support the interstice entrapment assumption. There was no systematic difference in binding between the two types of exocellulase CBMs--one that hydrolyzes cellulose from the nonreducing (CBM(Cel6B)) end and one that hydrolyzes cellulose from the reducing end (CBM(Cel48A)).     

Binding and reversibility of Thermobifida fusca Cel5A,Cel6B,and Cel48A and their respective catalytic domains to bacterial microcrystalline cellulose

The binding and reversibility of Thermobifida fusca intact Cel5A, Cel5B, and Cel48A and their corresponding catalytic domains (CDs) to bacterial microcrystalline cellulose (BMCC) were studied at 5 degrees C. The binding of the intact cellulases and of corresponding CDs to BMCC was irreversible in all regions: Langmuir binding (region I), interstice penetration (region II), and interstice saturation (region III). The three cellulose binding domains (CBMs) bind reversibly in "region I" although their respective CDs do not. The irreversible binding of these enzymes in the Langmuir region does not satisfy the Langmuir assumption; however, the overall fit of the Interstice Saturation model, which includes binding in MBCC interstices as well as on the freely accessible surface (Jung et al., 2002a) is good. The main limitation of the model is that it does not explicitly address a mechanism for forming the enzyme-substrate complex within the active site of the CDs.     

Kinetic studies of Thermobifida fusca Cel9A active site mutant enzymes

Thermobifida fusca Cel9A-90, an unusual family 9 enzyme, is a processive endoglucanase containing a catalytic domain closely linked to a family 3c cellulose binding domain (Cel9A-68) followed by a fibronectin III-like domain and a family 2 cellulose binding domain. To study its catalytic mechanism, 12 mutant genes with changes in five conserved residues of Cel9A-68 were constructed, cloned, and expressed in Escherichia coli. The purified mutant enzymes were assayed for their activities on (carboxymethyl)cellulose, phosphoric acid-swollen cellulose, bacterial microcrystalline cellulose, and 2,4-dinitrophenyl beta-D-cellobioside. They were also tested for ligand binding, enzyme processivity, and thermostability. The results clearly show that E424 functions as the catalytic acid, D55 and D58 are both required for catalytic base activity, and Y206 plays an important role in binding, catalysis, and processivity, while Y318 plays an important role in binding of crystalline cellulose substrates and is required for processivity. Several amino acids located in a loop at the end of the catalytic cleft (T245-L251) were deleted from Cel9A-68, and this enzyme showed slightly improved filter paper activity and binding to BMCC but otherwise behaved like the wild-type enzyme. The FnIII-like domain was deleted from Cel9A-90, reducing BMCC activity to 43% of the wild type.     

Interaction between the CBM of Cel9A from Thermobifida fusca and cellulose fibers

Molecular docking and molecular dynamics (MD) simulations were used to investigate the binding of a cellodextrin chain in a crystal-like conformation to the carbohydrate-binding module (CBM) of Cel9A from Thermobifida fusca. The fiber was found to bind to the CBM in a single and well-defined configuration in-line with the catalytic cleft, supporting the hypothesis that this CBM plays a role in the catalysis by feeding the catalytic domain (CD) with a polysaccharide chain. The results also expand the current known list of residues involved in the binding. The polysaccharide-protein attachment is shown to be mediated by five amine/amide-containing residues. E478 and E559 were found not to interact directly with the sugar chain; instead they seem to be responsible to stabilize the binding motif via hydrogen bonds.     

Computational and experimental studies of the catalytic mechanism of Thermobifida fusca cellulase Cel6A (E2)

Mutagenesis experiments suggest that Asp79 in cellulase Cel6A (E2) from Thermobifida fusca has a catalytic role, in spite of the fact that this residue is more than 13 A from the scissile bond in models of the enzyme-substrate complex built upon the crystal structure of the protein. This suggests that there is a substantial conformational shift in the protein upon substrate binding. Molecular mechanics simulations were used to investigate possible alternate conformations of the protein bound to a tetrasaccharide substrate, primarily involving shifts of the loop containing Asp79, and to model the role of water in the active site complex for both the native conformation and alternative low-energy conformations. Several alternative conformations of reasonable energy have been identified, including one in which the overall energy of the enzyme-substrate complex in solution is lower than that of the conformation in the crystal structure. This conformation was found to be stable in molecular dynamics simulations with a cellotetraose substrate and water. In simulations of the substrate complexed with the native protein conformation, the sugar ring in the -1 binding site was observed to make a spontaneous transition from the (4)C(1) conformation to a twist-boat conformer, consistent with generally accepted glycosidase mechanisms. Also, from these simulations Tyr73 and Arg78 were found to have important roles in the active site. Based on the results of these various MD simulations, a new catalytic mechanism is proposed. Using this mechanism, predictions about the effects of changes in Arg78 were made which were confirmed by site-directed mutagenesis.     

Cloning, expression and characterization of a family 48 exocellulase, Cel48A, from Thermobifida fusca.

The gene for a 104-kDa exocellulase, Cel48A, formerly E6, was cloned from Thermobifida fusca into Escherichia coli and Streptomyces lividans. The DNA sequence revealed a type II cellulose-binding domain at the N-terminus, followed by a FNIII-like domain and ending with a glycosyl hydrolase Family 48 catalytic domain. The enzyme and catalytic domain alone were each expressed in and purified from S. lividans and had very low catalytic activity on swollen cellulose, carboxymethyl cellulose, bacterial microcrystalline cellulose and filter paper. However, in synergistic assays on filter paper, the addition of Cel48A to a balanced mixture of T. fusca endocellulase and exocellulase increased the specific activity from 7.9 to 11.7 micromol cellobiose.min-1.mL-1, more than 15-fold higher than any single enzyme alone. Cel48A retained > 50% of its maximum activity from pH 5 to 9 and from 40 to 60 degrees C. Using SWISSMODEL, the amino-acid sequence of the Cel48Acd was modeled to the known structure of Clostridium cellulolyticum CelF. Family 48 enzymes are remarkably homologous at 35% identity for all their catalytic domains and some of the properties of the 10 members are discussed.     

Purification and characterization of Thermobifida fusca xylanase 10B

Thermobifida fusca grows well on cellulose and xylan, and produces a number of cellulases and xylanases. The gene encoding a previously unstudied endoxylanase, xyl10B, was overexpressed in E. coli, and the protein was purified and characterized. Mature Xyl10B is a 43-kDa glycohydrolase with a short basic domain at the C-terminus. It has moderate thermostability, maintaining 50% of its activity after incubation for 16 h at 62 degrees C, and is most active between pH 5 and 8. Xyl10B is produced by growth of T. fusca on xylan or Solka Floc but not on pure cellulose. Mass spectroscopic analysis showed that Xyl10B produces xylobiose as the major product from birchwood and oat spelts xylan and that its hydrolysis products differ from those of T. fusca Xyl11A. Xyl10B hydrolyzes various p-nitrophenyl-sugars, including p-nitrophenyl alpha-D-arabinofuranoside, p-nitrophenyl-beta-D-xylobioside, p-nitrophenyl-beta-D-xyloside, and p-nitrophenyl-beta-D-cellobioside. Xyl11A has higher activity on xylan substrates, but Xyl10B produced more reducing sugars from corn fiber than did Xyl11A.     

Thermobifida fusca family-6 cellulases as potential designer cellulosome components

During the course of our studies on the structure-function relationship of cellulosomes, we were interested in converting the free cellulase system of the aerobic bacterium, Thermobifida fusca, to a cellulosomal system. For this purpose, the cellulose-binding modules (CBM) of two T. fusca family-6 cellulases, endoglucanase Cel6A and exoglucanase Cel6B, were replaced by divergent dockerin modules. Thus far, family-6 cellulases have not been shown to be members of natural cellulosome systems. The resultant chimaeric proteins, 6A-c and t-6B, respectively, were purified and found to interact specifically and stoichiometrically with their corresponding cohesin modules, indicating their suitability for use as components in 'designer cellulosomes'. Both chimaeric enzymes showed somewhat decreased but measurable levels of activity on carboxymethyl cellulose, consistent with the known endo- and exo-glucanase character of the parent enzymes. The activity of 6A-c on phosphoric acid swollen cellulose was also consistent with that of the wild-type endoglucanase Cel6A. The startling finding of the present research was the extent of degradation of this substrate by the chimaeric enzyme t-6B. Wild-type exoglucanase Cel6B exhibited very low activity on this substrate, while the specific activity of t-6B was 14-fold higher than the parent enzyme.     

Synergistic activity of Paenibacillus sp. BP-23 cellobiohydrolase Cel48C in association with the contiguous endoglucanase Cel9B and with endo- or exo-acting glucanases from Thermobifida fusca

Cellobiohydrolase Cel48C from Paenibacillus sp. BP-23, an enzyme displaying limited activity on most cellulosic substrates, was assayed for activity in the presence of other bacterial endo- or exocellulases. Significant enhanced activity was observed when Cel48C was incubated in the presence of Paenibacillus sp. BP-23 endoglucanase Cel9B or Thermobifida fusca cellulases Cel6A and Cel6B, indicating that Cel48C acts synergistically with them. Maximum synergism rates on bacterial microcrystalline cellulose or filter paper were obtained with a mixture of Paenibacillus cellulases Cel9B and Cel48C, accompanied by T. fusca exocellulase Cel6B. Synergism was also observed in cell extracts from recombinant clone E. coli pUCel9-Cel48 expressing the two contiguous Paenibacillus cellulases Cel9B and Cel48C. The enhanced cellulolytic activity displayed by the cellulase mixtures assayed could be used as an efficient tool for biotechnological applications like pulp and paper manufacturing.     

Thermobifida fusca family-6 cellulases as potential designer cellulosome components

During the course of our studies on the structure–function relationship of cellulosomes, we were interested in converting the free cellulase system of the aerobic bacterium, Thermobifida fusca, to a cellulosomal system. For this purpose, the cellulose-binding modules (CBM) of two T. fusca family-6 cellulases, endoglucanase Cel6A and exoglucanase Cel6B, were replaced by divergent dockerin modules. Thus far, family-6 cellulases have not been shown to be members of natural cellulosome systems. The resultant chimaeric proteins, 6A-c and t-6B, respectively, were purified and found to interact specifically and stoichiometrically with their corresponding cohesin modules, indicating their suitability for use as components in ‘designer cellulosomes’. Both chimaeric enzymes showed somewhat decreased but measurable levels of activity on carboxymethyl cellulose, consistent with the known endo- and exo-glucanase character of the parent enzymes. The activity of 6A-c on phosphoric acid swollen cellulose was also consistent with that of the wild-type endoglucanase Cel6A. The startling finding of the present research was the extent of degradation of this substrate by the chimaeric enzyme t-6B. Wild-type exoglucanase Cel6B exhibited very low activity on this substrate, while the specific activity of t-6B was 14-fold higher than the parent enzyme.     

Determination of the molecular states of the processive endocellulase Thermobifida fusca Cel9A during crystalline cellulose depolymerization

Detailed understanding of cell wall degrading enzymes is important for their modeling and industrial applications, including in the production of biofuels. Here we used Cel9A, a processive endocellulase from Thermobifida fusca, to demonstrate that cellulases that contain a catalytic domain (CD) attached to a cellulose binding module (CBM) by a flexible linker exist in three distinct molecular states. By measuring the ability of a soluble competitor to reduce Cel9A activity on an insoluble substrate, we show that the most common state of Cel9A is bound via its CBM, but with its CD unoccupied by the insoluble substrate. These findings are relevant for kinetic modeling and microscopy studies of modular glycoside hydrolases.     

Crystal structure of Thermobifida fusca endoglucanase Cel6A in complex with substrate and inhibitor: the role of tyrosine Y73 in substrate ring distortion

Endoglucanase Cel6A from Thermobifida fusca hydrolyzes the beta-1,4 linkages in cellulose at accessible points along the polymer. The structure of the catalytic domain of Cel6A from T. fusca in complex with a nonhydrolysable substrate analogue that acts as an inhibitor, methylcellobiosyl-4-thio-beta-cellobioside (Glc(2)-S-Glc(2)), has been determined to 1.5 A resolution. The glycosyl unit in subsite -1 was sterically hindered by Tyr73 and forced into a distorted (2)S(o) conformation. In the enzyme where Tyr73 was mutated to a serine residue, the hindrance was removed and the glycosyl unit in subsite -1 had a relaxed (4)C(1) chair conformation. The relaxed conformation was seen in two complex structures of the mutated enzyme, with cellotetrose (Glc(4)) at 1.64 A and Glc(2)-S-Glc(2) at 1.04 A resolution.     

Cel6B of Thermobifidus fusca and a Cel5-CBM6 of Ruminococcus albus containing a cellulose binding site show synergistic effect on hydrolysis of native plant cellulose

We have established an improved large deletion method in Escherichia coli genome using a combination of two different recombination systems, lambda Red and Cre/loxP. The loxP site could be rapidly and efficiently integrated in the genome by lambda Red and large deletions of both 117- and 165-kbp regions could be generated in 100% efficiency by Cre/loxP. Comparative genomic hybridization microarray experiments of deletion strains indicated that deletions were generated only in expected regions of the genome. These results have demonstrated that the method is useful for genome engineering in E. coli.