Binding specificity and thermodynamics of a family 9 carbohydrate-binding module from Thermotoga maritima xylanase 10A.

The C-terminal family 9 carbohydrate-binding module of xylanase 10A from Thermotoga maritima (CBM9-2) binds to amorphous cellulose, crystalline cellulose, and the insoluble fraction of oat spelt xylan. The association constants (K(a)) for adsorption to insoluble polysaccharides are 1 x 10(5) to 3 x 10(5) M(-1). Of the soluble polysaccharides tested, CBM9-2 binds to barley beta-glucan, xyloglucan, and xylan. CBM9-2 binds specifically to the reducing ends of cellulose and soluble polysaccharides, a property that is currently unique to this CBM. CBM9-2 also binds glucose, xylose, galactose, arabinose, cellooligosaccharides, xylooligosaccharides, maltose, and lactose, with affinities ranging from 10(3) M(-1) for monosaccharides to 10(6) M(-1) for disaccharides and oligosaccharides. Cellooligosaccharides longer than two glucose units do not bind with improved affinity, indicating that cellobiose is sufficient to occupy the entire binding site. In general, the binding reaction is dominated by favorable changes in enthalpy, which are partially compensated by unfavorable entropy changes.

Rapid determination of adenoviral vector titers by quantitative real-time PCR.

Replication defective adenoviruses have been used as vectors in a variety of settings including gene transfer, gene manipulation, and functionality studies. A quantitative real-time PCR-based assay is described for rapid determination of physical titers of recombinant adenovirus vectors. This method is based on amplification of a 77 bp fragment located near the left end of the adenovirus type 5 genome. Evaluation of this method demonstrated that it is simple, sensitive and reproducible, and has a dynamic range of quantitation over 5 logs. This assay is applicable to purified adenovirus as well as vectors prepared by simple cell lysis procedure, requiring only a small amount of starting material. The simplicity and short turn-around time of this assay should facilitate rapid titer determination for a large collection of adenoviral vectors.

A novel mechanism of xylan binding by a lectin-like module from Streptomyces lividans xylanase 10A.

The C-terminal module of xylanase 10A from Streptomyces lividans is a family 13 carbohydrate-binding module (CBM13). CBM13 binds mono- and oligo-saccharides with association constants of approximately 1x10(2) M(-1)-1x10(3) M(-1). It appears to be specific only for pyranose sugars. CBM13 binds insoluble and soluble xylan, holocellulose, pachyman, lichenan, arabinogalactan and laminarin. The association constant for binding to soluble xylan is (6.2+/-0. 6)x10(3)/mol of xylan polymer. Site-directed mutation indicates the involvement of three functional sites on CBM13 in binding to soluble xylan. The sites are similar in sequence, and are predicted to have similar structures, to the alpha, beta and gamma sites of ricin toxin B-chain, which is also in family 13. The affinity of a single binding site on CBM13 for soluble xylan is only approximately (0. 5+/-0.1)x10(3)/mol of xylan. The binding of CBM13 to soluble xylan involves additive and co-operative interactions between the three binding sites. This mechanism of binding has not previously been reported for CBMs binding polysaccharides. CBM13 is the first bacterial module from family 13 to be described in detail.

Affinity electrophoresis for the identification and characterization of soluble sugar binding by carbohydrate-binding modules.

Affinity electrophoresis was used to identify and quantify the interaction of carbohydrate-binding modules (CBMs) with soluble polysaccharides. Association constants determined by AE were in excellent agreement with values obtained by isothermal titration calorimetry and fluorescence titration. The method was adapted to the identification, study and characterization of mutant carbohydrate-binding modules with altered affinities and specificities. Competition affinity electrophoresis was used to monitor binding of small, soluble mono- and disaccharides to one of the modules.

Binding site analysis of cellulose binding domain CBD(N1) from endoglucanse C of Cellulomonas fimi by site-directed mutagenesis.

Endoglucanase C (CenC), a beta1,4 glucanase from the soil bacterium Cellulomonas fimi, binds to amorphous cellulose via two homologous cellulose binding domains, termed CBD(N1) and CBD(N2). In this work, the contributions of 10 amino acids within the binding cleft of CBD(N1) were evaluated by single site-directed mutations to alanine residues. Each isolated domain containing a single mutation was analyzed for binding to an insoluble amorphous preparation of cellulose, phosphoric acid swollen Avicel (PASA), and to a soluble glucopyranoside polymer, barley beta-glucan. The effect of any given mutation on CBD binding was similar for both substrates, suggesting that the mechanism of binding to soluble and insoluble substrates is the same. Tyrosines 19 and 85 were essential for tight binding by CBD(N1) as their replacement by alanine results in affinity decrements of approximately 100-fold on PASA, barley beta-glucan, and soluble cellooligosaccharides. The tertiary structures of unbound Y19A and Y85A were assessed by heteronuclear single quantum coherence (HSQC) spectroscopy. These studies indicated that the structures of both mutants were perturbed but that all perturbations are very near to the site of mutation.

Structure and binding specificity of the second N-terminal cellulose-binding domain from Cellulomonas fimi endoglucanase C.

The 1,4-beta-glucanase CenC from Cellulomonas fimi contains two cellulose-binding domains, CBD(N1) and CBD(N2), arranged in tandem at its N-terminus. These homologous CBDs are distinct in their selectivity for binding amorphous and not crystalline cellulose. Multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy was used to determine the tertiary structure of CBD(N2) in the presence of saturating amounts of cellopentaose. A total of 1996 experimental restraints were used to calculate an ensemble of 21 final structures for the protein. CBD(Nu2) is composed of 11 beta-strands, folded into two antiparallel beta-sheets, with a topology of a jellyroll beta-sandwich. On the basis of patterns of chemical shift perturbations accompanying the addition of cellooligosaccharides, as well as the observation of intermolecular protein-sugar NOE interactions, the cellulose-binding site of CBD(N2) was identified as a cleft that lies across one face of the beta-sandwich. The thermodynamic basis for the binding of cellooligosaccharides was investigated using isothermal titration calorimetry and NMR spectroscopy. Binding is enthalpically driven and consistent with a structural model involving hydrogen bonding between the equatorial hydroxyls of the glucopyranosyl rings and polar amino acid side chains lining the CBD(N2) cleft. Affinity electrophoresis was used to determine that CBD(N2) also binds soluble beta-1,4-linked polymers of glucose, including hydroxyethylcellulose and beta-1,3-1,4-glucans. This study complements a previous analysis of CBD(N1) [Johnson, P. E., Joshi, M. D., Tomme, P., Kilburn, D. G., and McIntosh, L. P. (1996) Biochemistry 35, 14381-14394] and demonstrates that the homologous CBDs from CenC share very similar structures and sugar binding properties.

Characterization and affinity applications of cellulose-binding domains.

Cellulose-binding domains (CBDs) are discrete protein modules found in a large number of carbohydrolases and a few nonhydrolytic proteins. To date, almost 200 sequences can be classified in 13 different families with distinctly different properties. CBDs vary in size from 4 to 20 kDa and occur at different positions within the polypeptides; N-terminal, C-terminal and internal. They have a moderately high and specific affinity for insoluble or soluble cellulosics with dissociation constants in the low micromolar range. Some CBDs bind irreversibly to cellulose and can be used for applications involving immobilization, others bind reversibly and are more useful for separations and purifications. Dependent on the CBD used, desorption from the matrix can be promoted under various different conditions including denaturants (urea, high pH), water, or specific competitive ligands (e.g. cellobiose). Family I and IV CBDs bind reversibly to cellulose in contrast to family II and III CBDs which are in general, irreversibly bound. The binding of family II CBDs (CBD(Cex)) to crystalline cellulose is characterized by a large favourable increase in entropy indicating that dehydration of the sorbent and the protein are the major driving forces for binding. In contrast, binding of family IV CBDs (CBD(N1)) to amorphous or soluble cellulosics is driven by a favourable change in enthalpy which is partially offset by an unfavourable entropy change. Hydrogen bond formation and van der Waals interactions are the main driving forces for binding. CBDs with affinity for crystalline cellulose are useful tags for classical column affinity chromatography. The affinity of CBD(N1) for soluble cellulosics makes it suitable for use in large-scale aqueous two-phase affinity partitioning systems.

Calcium binding by the N-terminal cellulose-binding domain from Cellulomonas fimi beta-1,4-glucanase CenC.

The interaction of the N-terminal cellulose-binding domain, CBDN1, from Cellulomonas fimi beta-1,4-glucanase CenC with calcium was investigated using NMR spectroscopy and calorimetry. CBDN1 binds a single calcium ion with an equilibrium association constant of approximately 10(5) M-1 at 35 degreesC and pH 6.0. Binding is exothermic (-42 +/- 2 kJ mol-1) under these conditions and is accompanied by a small negative change in heat capacity (DeltaCp = -0.41 +/- 0.16 kJ mol-1 K-1). From an NMR line shape analysis, the rate constants for calcium association and dissociation were found to be (5 +/- 2) x 10(7) s-1 M-1 and (4.5 +/- 0.6) x 10(2) s-1, respectively. The rapid association kinetics indicate that the calcium-binding site on CBDN1 is accessible and, to the first approximation, preformed. Based on patterns of chemical shift perturbations, and structural comparisons with the Bacillus sp. 1, 3-1,4-beta-glucanases, the backbone carbonyl oxygens of Thr8, Gly30, and Asp142 and a side chain carboxyl oxygen of Asp142 are postulated to form the calcium-binding site of CBDN1. Consistent with the calcium-independent affinity of CBDN1 for cellopentaose, this exposed site is located on the face of CBDN1 opposite to that forming the oligosaccharide-binding cleft. The midpoint denaturation temperature of CBDN1 is increased by approximately 8 degreesC at pH 6.0 in the presence of saturating amounts of calcium, confirming that metal ion binding is thermodynamically linked to native-state stability.

Stability and oligosaccharide binding of the N1 cellulose-binding domain of Cellulomonas fimi endoglucanase CenC.

Differential scanning calorimetry has been used to study the thermal stability and oligosaccharide-binding thermodynamics of the N-terminal cellulose-binding domain of Cellulomonas fimi beta-1,4-glucanase CenC (CBDN1). CBDN1 has a relatively low maximum stability (delta Gmax = 33 kJ/mol = 216 J/residue at 1 degree C and pH 6.1) compared to other small single-domain globular proteins. The unfolding is fully reversible between pH 5.5 and 9 and in accordance with the two-state equilibrium model between pH 5.5 and 11. When the single disulfide bond in CBDN1 is reduced, the protein remains unfolded at all conditions, as judged by NMR spectroscopy. This indicates that the intramolecular cross-link makes a major contribution to the stability of CBDN1. The measured heat capacity change of unfolding (delta Cp = 7.5 kJ mol-1 K-1) agrees well with that calculated from the predicted changes in the solvent accessible nonpolar and polar surface areas upon unfolding. Extrapolation of the specific enthalpy and entropy of unfolding to their respective convergence temperature indicates that per residue unfolding energies for CBDN1, an isolated domain, are in accordance with those found by Privalov (1) for many single-domain globular proteins. DSC thermograms of the unfolding of CBDN1 in the presence of various concentrations of cellopentaose were fit to a thermodynamic model describing the linkage between protein-ligand binding and protein unfolding. A global two-dimensional minimization routine is used to regress the binding enthalpy, binding constant, and unfolding thermodynamics for the CBDN1-cellopentaose system. Extrapolated binding constants are in quantitative agreement with those determined by isothermal titration calorimetry at 35 degrees C.

Structure of the N-terminal cellulose-binding domain of Cellulomonas fimi CenC determined by nuclear magnetic resonance spectroscopy.

Multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy was used to determine the tertiary structure of the 152 amino acid N-terminal cellulose-binding domain from Cellulomonas fimi 1,4-beta-glucanase CenC (CBDN1). CBDN1 was studied in the presence of saturating concentrations of cellotetraose, but due to spectral overlap, the oligosaccharide was not included in the structure calculations. A total of 1705 interproton nuclear Overhauser effect (NOE), 56 phi, 88 psi, 42 chi 1, 9 chi 2 dihedral angle, and 88 hydrogen-bond restraints were used to calculate 25 final structures. These structures have a rmsd from the average of 0.79 +/- 0.11 A for all backbone atoms excluding disordered termini and 0.44 +/- 0.05 A for residues with regular secondary structures. CBDN1 is composed of 10 beta-strands, folded into two antiparallel beta-sheets with the topology of a jelly-roll beta-sandwich. The strands forming the face of the protein previously determined by chemical shift perturbations to be responsible for cellooligosaccharide binding [Johnson, P. E., Tomme, P., Joshi, M. D., & McIntosh, L. P. (1996) Biochemistry 35, 13895-13906] are shorter than those forming the opposite side of the protein. This results in a 5-stranded binding cleft, containing a central strip of hydrophobic residues that is flanked on both sides by polar hydrogen-bonding groups. The presence of this cleft provides a structural explanation for the unique selectivity of CBDN1 for amorphous cellulose and other soluble oligosaccharides and the lack of binding to crystalline cellulose. The tertiary structure of CBDN1 is strikingly similar to that of the bacterial 1,3-1,4-beta-glucanases, as well as other sugar-binding proteins with jelly-roll folds.

Interaction of polysaccharides with the N-terminal cellulose-binding domain of Cellulomonas fimi CenC. 1. Binding specificity and calorimetric analysis.

The carbohydrate-binding specificity of the N-terminal cellulose-binding domain (CBDN1) from Cellulomonas fimi beta-1,4-glucanase C (CenC) was investigated using affinity electrophoresis, binding assays and microcalorimetry in parallel with NMR and difference ultraviolet absorbance spectroscopy [Johnson, P.E., Tomme, P., Joshi, M.D., & McIntosh, I., P. (1996) Biochemistry 35, 13895-13906]. Binding of CBDN1 on insoluble cellulose is distinctly different from other cellulose-binding domains. CBDN1 binds amorphous cellulose (phosphoric acid-swollen) with high affinity (Kr = 5.1 L g-1), binds Avicel weakly and does not bind highly crystalline bacterial or tunicin cellulose. Moreover, CBDN1 binds soluble cellooligosaccharides and beta-1,4-linked oligomers of glucose such as hydroxyethycellulose, soluble beta-1,3-1,4-glucans from barley and oat, but has no affinity for alpha-1,4-, beta-1,3-, or beta-1,6-polymers of glucose. This is the first report of a cellulose-binding domain with strong and specific affinity for soluble glycans. The thermodynamics for binding of CBDN1 to oligosaccharides, soluble glycans, and phosphoric acid-swollen cellulose were investigated by titration microcalorimetry. At least four beta-1,4-linked glucopyranosides are required to detect binding. For larger glucans, with five or more glucopyranoside units, the binding constants and standard free energy changes are virtually independent of the glucan chain length, indicating that cellopentaose completely fills the binding site. Binding is moderately strong with binding constants ranging from 3,200 +/- 500 M-1 for cellotetraose, to 25,000 +/- 3,000 M-1 for the larger sugars. The reactions are controlled by favorable standard free enthalpy changes which are compensated in a linear fashion by a significant decrease in entropy. A predominance of polar interactions such as hydrogen bonding together with van der Waals interactions provide the major driving forces for the binding event.

Interaction of soluble cellooligosaccharides with the N-terminal cellulose-binding domain of Cellulomonas fimi CenC 2. NMR and ultraviolet absorption spectroscopy.

The N-terminal cellulose-binding domain (CBDN1) from Cellulomonas fimi beta-1,4-glucanase CenC binds amorphous but not crystalline cellulose. To investigate the structural and thermodynamic bases of cellulose binding, NMR and difference ultraviolet absorbance spectroscopy were used in parallel with calorimetry (Tomme, P., Creagh, A. L., Kilburn, D. G., & Haynes, C. A., (1996) Biochemistry 35, 13885-13894) to characterize the interaction of soluble cellooligosaccharides with CBDN1. Association constants, determined from the dependence of the amide 1H and 15N chemical shifts of CBDN1 upon added sugar, increase from 180 +/- 60 M-1 for cellotriose to 4,200 +/- 720 M-1 for cellotetraose, 34,000 +/- 7,600 M-1 for cellopentaose, and an estimate of 50,000 M-1 for cellohexaose. This implies that the CBDN1 cellulose-binding site spans approximately five glucosyl units. On the basis of the observed patterns of amide chemical shift changes, the cellooligosaccharides bind along a five-stranded beta-sheet that forms a concave face of the jelly-roll beta-sandwich structure of CBDN1. This beta-sheet contains a strip of hydrophobic side chains flanked on both sides by polar residues. NMR and difference ultraviolet absorbance measurements also demonstrate that tyrosine, but not tryptophan, side chains may be involved in oligosaccharide binding. These results lead to a model in which CBDN1 interacts with soluble cellooligosaccharides and, by inference, with single polysaccharide chains in regions of amorphous cellulose, primarily through hydrogen bonding to the equatorial hydroxyl groups of the pyranose rings. Van der Waals stacking of the sugar rings against the apolar side chains may augment binding. CBDN1 stands in marked contrast to previously characterized CBDs that absorb to crystalline cellulose via a flat binding surface dominated by exposed aromatic rings.

Characterization of CenC, an enzyme from Cellulomonas fimi with both endo- and exoglucanase activities.

The cenC gene, encoding beta-1,4-glucanase C (CenC) from Cellulomonas fimi, was overexpressed in Escherichia coli with a tac-based expression vector. The resulting polypeptide, with an apparent molecular mass of 130 kDa, was purified from the cell extracts by affinity chromatography on cellulose followed by anion-exchange chromatography. N-terminal sequence analysis showed the enzyme to be properly processed. Mature CenC was optimally active at pH 5.0 and 45 degrees C. The enzyme was extremely active on soluble, fluorophoric, and chromophoric glycosides (4-methylumbelliferyl beta-glycosides, 2'-chloro-4'-nitrophenyl-beta-D-cellobioside, and 2'-chloro-4'-nitrophenyl-lactoside) and efficiently hydrolyzed carboxymethyl cellulose, barley beta-glucan, lichenan, and, to a lesser extent, glucomannan. CenC also hydrolyzed acid-swollen cellulose, Avicel, and bacterial microcrystalline cellulose. However, degradation of the latter was slow compared with its degradation by CenB, another C. fimi cellulose belonging to the same enzyme family. CenC acted with inversion of configuration at the anomeric carbon, in accordance with its classification as a family 9 member. The enzyme released mainly cellobiose from soluble cellodextrins and insoluble cellulose. Attack appeared to be from the reducing chain ends. Analysis of carboxymethyl cellulose hydrolysis suggests that CenC is semiprocessive enzyme with both endo- and exoglucanase activities.

Comparison of a fungal (family I) and bacterial (family II) cellulose-binding domain.

A family II cellulose-binding domain (CBD) of an exoglucanase/xylanase (Cex) from the bacterium Cellulomonas fimi was replaced with the family I CBD of cellobiohydrolase I (CbhI) from the fungus Trichoderma reesei. Expression of the hybrid gene in Escherichia coli yielded up to 50 mg of the hybrid protein, CexCBDCbhI, per liter of culture supernatant. The hybrid was purified to homogeneity by affinity chromatography on cellulose. The relative association constants (Kr) for the binding of Cex, CexCBDCbhI, the catalytic domain of Cex (p33), and CbhI to bacterial microcrystalline cellulose (BMCC) were 14.9, 7.8, 0.8, and 10.6 liters g-1, respectively. Cex and CexCBDCbhI had similar substrate specificities and similar activities on crystalline and amorphous cellulose. Both released predominantly cellobiose and cellotriose from amorphous cellulose. CexCBDCbhI was two to three times less active than Cex on BMCC, but significantly more active than Cex on soluble cellulose and on xylan. Unlike Cex, the hybrid protein neither bound to alpha-chitin nor released small particles from dewaxed cotton fibers.

Enhancement of the endo-beta-1,4-glucanase activity of an exocellobiohydrolase by deletion of a surface loop.

In the commonly accepted mechanism for enzymatic hydrolysis of cellulose, endo-beta-1,4-glucanases randomly cleave glucosidic bonds within glucan polymers, providing sites for attack by exo-cellobiohydrolases (EC 3.2.1.91). It has been proposed that hydrolysis by Trichoderma reesei cellobiohydrolase II is restricted to the ends of cellulose polymers because two surface loops cover its active site to form a tunnel. In a closely related endoglucanase, E2 from Thermomonospora fusca, access to the substrate appears to be relatively unhindered because the carboxyl-proximal loop is shortened, and the amino-proximal loop is displaced. The hypothesis was examined by deletion of a region in Cellulomonas fimi cellobiohydrolase A corresponding to part of the carboxyl-proximal loop of T. reesei cellobiohydrolase II. The mutation enhanced the endoglucanase activity of the enzyme on soluble O-(carboxymethyl)cellulose and altered its activities on 2',4'-dinitrophenyl-beta-D-cellobioside, insoluble cellulose, and cellotetraose.

Stereochemical course of hydrolysis catalysed by Cellulomonas fimi CenE, a member of a new family of beta-1,4-glucanases.

The gene for a previously identified, extracellular, 120 kDa cellulose-binding protein (Cbp120) was isolated from a Cellulomonas fimi genomic library and expressed in Escherichia coli. Qualitative analysis of CM-cellulose hydrolysis shows that Cbp120 is an endo-beta-1,4-glucanase. Cbp120, now renamed CenE, catalyzes hydrolysis of cellohexaose with inversion of anomeric carbon configuration, characteristic of a single displacement reaction. Partial sequencing of its gene shows that CenE has significant sequence similarity with the catalytic domains of five enzymes from cellulolytic bacteria. It is proposed that the six enzymes form a new family of beta-1,4-glucanases. CenE is the first enzyme from this family to be characterized stereochemically.

An internal cellulose-binding domain mediates adsorption of an engineered bifunctional xylanase/cellulase.

A chimeric xylanase/endoglucanase (XynCenA) with an internal cellulose-binding domain was constructed by fusing the Bacillus subtilis xyn gene fragment to the 5'-end of the Cellulomonas fimi cenA. A polyhistidine-encoding sequence was also fused to the 5'-end of the xyn gene. The gene fusion was overexpressed in Escherichia coli and the fusion polypeptide purified from the cell extracts using the polyhistidine tail. The hybrid protein behaved like the parental endoglucanase or xylanase when assayed on a number of soluble and insoluble cellulosic substrates or xylans. The presence of two distinct active sites and the internal cellulose-binding domain did not significantly affect the hydrolysis of any of these substrates. However, the fusion protein exhibited a strong affinity for both microcrystalline cellulose (Avicel) and regenerated chitin. Like the parental endoglucanase, bound XynCenA could not be eluted from these polysaccharides with either low or high salt buffer or distilled water. More stringent conditions, such as 1% SDS or 8 M guanidinium hydrochloride, fully desorbed the protein. The fusion protein did not adsorb significantly to insoluble xylan.

Mode of action of endoglucanase III from Trichoderma reesei.

Endoglucanase III (EG III) was purified to homogeneity from the culture medium of Trichoderma reesei QM 9414. It has a molecular mass of 48 kDa, and an isoelectric point of 5.1. Maximal activity was observed between pH4 and 5. Celloligosaccharides and their chromophoric derivatives were used as substrates, and the reaction products were analysed by quantitative h.p.l.c. Nucleophilic competition experiments (between methanol and water) allowed unequivocal assessment of cleavage sites. EG III preferentially released cellobiose (or the corresponding glycoside) from the reducing end of the higher cellodextrins. A putative binding model containing five subsites is proposed. The pH-dependence of 4'-methylumbelliferyl beta-cellotrioside hydrolysis indicates the presence of a protonated group with a pK 5.5 in the reaction mechanism, and the possible involvement of a carboxy group is corroborated by a temperature study (delta Hion = -15.9 J/mol). This, together with independent evidence from affinity-labelling experiments [Tomme, Macarrón and Claeyssens (1991) Cellulose '91, New Orleans, Abstr. 32] and n.m.r. studies [Gebbler, Gilkes, Claeyssens, Wilson, Béguin, Wakarchuk, Kilburn, Miller, Warren and Withers (1992) J. Biol. Chem. 267, 12559-12561], favours the assumption of a lysozyme-type (retention of configuration, two essential carboxy groups) mechanism for this family A cellulase.