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N R Gilkes

Publications and source records attributed to N R Gilkes.

At least 19 recordsLinked to original sources
PubMed2001

Do cellulose binding domains increase substrate accessibility?

This article provides an overview of various theories proposed during the past five decades to describe the enzymatic hydrolysis of cellulose highlighting the major shifts that these theories have undergone. It also describes the effect of the cellulose-binding domain (CBD) of an exoglucanase/xylanase from bacterium Cellulomonas fimi on the enzymatic hydrolysis of Avicel. Pretreatment of Avicel with CBDCex at 4 and 37 degrees C as well as simultaneous addition of CBDCex to the hydrolytic enzyme (Celluclast, Novo, Nordisk) reduced the initial rate of hydrolysis owing to irreversible binding of CBD proteins to the substrate's binding sites. Nonetheless, near complete hydrolysis was achieved even in the presence of CBDCex. Protease treatment of both pure and CBDCex-treated Avicel reduced the substrates' hydrolyzability, perhaps owing to proteolysis of the hydrolyzing enzyme (Celluclast) by the residual Proteinase K remaining in the substrate. Better protocols for complete removal of CBD proteins from the substrate need to be developed to investigate the effect of CBD adsorption on cellulose digestibility.

Binding Sites↗
PubMed2000

Analysis of binding of the family 2a carbohydrate-binding module from Cellulomonas fimi xylanase 10A to cellulose: specificity and identification of functionally important amino acid residues.

The family 2a carbohydrate-binding module (CBM2a) of xylanase 10A from Cellulomonas fimi binds to the crystalline regions of cellulose. It does not share binding sites with the N-terminal family 4 binding module (CBM4-1) from the cellulase 9B from C.fimi, a module that binds strictly to soluble sugars and amorphous cellulose. The binding of CBM2a to crystalline matrices is mediated by several residues on the binding face, including three prominent, solvent-exposed tryptophan residues. Binding to crystalline cellulose was analyzed by making a series of conservative (phenylalanine and tyrosine) and non-conservative substitutions (alanine) of each solvent-exposed tryptophan (W17, W54 and W72). Other residues on the binding face with hydrogen bonding potential were substituted with alanine. Each tryptophan plays a different role in binding; a tryptophan is essential at position 54, a tyrosine or tryptophan at position 17 and any aromatic residue at position 72. Other residues on the binding face, with the exception of N15, are not essential determinants of binding affinity. Given the specificity of CBM2a, the structure of crystalline cellulose and the dynamic nature of the binding of CBM2a, we propose a model for the interaction between the polypeptide and the crystalline surface.

Actinomycetales↗
PubMed1999

Comparing the thermodynamic stabilities of a related thermophilic and mesophilic enzyme.

Several models have been proposed to explain the high temperatures required to denature enzymes from thermophilic organisms; some involve greater maximum thermodynamic stability for the thermophile, and others do not. To test these models, we reversibly melted two analogous protein domains in a two-state manner. E2cd is the isolated catalytic domain of cellulase E2 from the thermophile Thermomonospora fusca. CenAP30 is the analogous domain of the cellulase CenA from the mesophile Cellulomonas fimi. When reversibly denatured in a common buffer, the thermophilic enzyme E2cd had a temperature of melting (Tm) of 72.2 degrees C, a van't Hoff enthalpy of unfolding (DeltaHVH) of 190 kcal/mol, and an entropy of unfolding (DeltaSu) of 0.55 kcal/(mol*K); the mesophilic enzyme CenAP30 had a Tm of 56.4 degrees C, a DeltaHVH of 107 kcal/mol, and a DeltaSu of 0. 32 kcal/(mol*K). The higher DeltaHVH and DeltaSu values for E2cd suggest that its free energy of unfolding (DeltaGu) has a steeper dependence on temperature at the Tm than CenAP30. This result supports models that predict a greater maximum thermodynamic stability for thermophilic enzymes than for their mesophilic counterparts. This was further explored by urea denaturation. Under reducing conditions at 30 degrees C, E2cd had a concentration of melting (Cm) of 5.2 M and a DeltaGu of 11.2 kcal/mol; CenAP30 had a Cm of 2.6 M and a DeltaGu of 4.3 kcal/mol. Under nonreducing conditions, the Cm and DeltaGu of CenAP30 were increased to 4.5 M and 10.8 kcal/mol at 30 degrees C; the Cm for E2cd was increased to at least 7.4 M at 32 degrees C. We were unable to determine a DeltaGu value for E2cd under nonreducing conditions due to problems with reversibility. These data suggest that E2cd attains its greater thermal stability (DeltaTm = 15.8 degrees C) through a greater thermodynamic stability (DeltaDeltaGu = 6.9 kcal/mol) compared to its mesophilic analogue CenAP30.

Actinomycetales↗
PubMed1998

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.

Binding Sites↗
PubMed1998

Analysis of molecular size distributions of cellulose molecules during hydrolysis of cellulose by recombinant Cellulomonas fimi beta-1,4-glucanases.

Four beta-1,4-glucanases (cellulases) of the cellulolytic bacterium Cellulomonas fimi were purified from Escherichia coli cells transformed with recombinant plasmids. Previous analyses using soluble substrates had suggested that CenA and CenC were endoglucanases while CbhA and CbhB resembled the exo-acting cellobiohydrolases produced by cellulolytic fungi. Analysis of molecular size distributions during cellulose hydrolysis by the individual enzymes confirmed these preliminary findings and provided further evidence that endoglucanase CenC has a more processive hydrolytic activity than CenA. The significant differences between the size distributions obtained during hydrolysis of bacterial microcrystalline cellulose and acid-swollen cellulose can be explained in terms of the accessibility of beta-1,4-glucan chains to enzyme attack. Endoglucanases and cellobiohydrolases were much more easily distinguished when the acid-swollen substrate was used.

Actinomycetales↗
PubMed1996

Probing the role of tryptophan residues in a cellulose-binding domain by chemical modification.

The cellulose-binding domain (CBDCex) of the mixed function glucanase-xylanase Cex from Cellulomonas fimi contains five tryptophans, two of which are located within the beta-barrel structure and three exposed on the surface (Xu GY et al., 1995, Biochemistry 34:6993-7009). Although all five tryptophans can be oxidized by N-bromosuccinimide (NBS), stopped-flow measurements show that three tryptophans react faster than the other two. NMR analysis during the titration of CBDCex with NBS shows that the tryptophans on the surface of the protein are fully oxidized before there is significant reaction with the two buried tryptophans. Additionally, modification of the exposed tryptophans does not affect the conformation of the backbone of CBDCex, whereas complete oxidation of all five tryptophans denatures the polypeptide. The modification of the equivalent of one and two tryptophans by NBS reduces binding of CBDCex to cellulose by 70% and 90%, respectively. This confirms the direct role of the exposed aromatic residues in the binding of CBDCex to cellulose. Although adsorption to cellulose does afford some protection against NBS, as evidenced by the increased quantity of NBS required to oxidize all of the tryptophan residues, the polypeptide can still be oxidized completely when adsorbed. This suggests that, whereas the binding appears to be irreversible overall [Ong E et al., 1989, Bio/Technology 7:604-607], each of the exposed tryptophans interacts reversibly with cellulose.

Binding Sites↗
PubMed1996

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.

Base Sequence↗
PubMed1995

Cellobiohydrolase B, a second exo-cellobiohydrolase from the cellulolytic bacterium Cellulomonas fimi.

The gene cbhB from the cellulolytic bacterium Cellulomonas fimi encodes a polypeptide of 1090 amino acids. Cellobiohydrolase B (CbhB) is 1037 amino acids long, with a calculated molecular mass of 109765 Da. The enzyme comprises five domains: an N-terminal catalytic domain of 643 amino acids, three fibronectin type III repeats of 97 amino acids each, and a C-terminal cellulose-binding domain of 104 amino acids. The catalytic domain belongs to family 48 of glycosyl hydrolases. CbhB has a very low activity on CM-cellulose. Viscometric analysis of CM-cellulose hydrolysis indicates that the enzyme is an exoglucanase. Cellobiose is the major product of hydrolysis of cellulose. In common with two other exoglycanases from C. fimi, CbhB has low but detectable endoglucanase activity. CbhB is the second exo-cellobiohydrolase found in C. fimi. Therefore, the cellulase system of C. fimi resembles those of fungi in comprising multiple endoglucanases and cellobiohydrolases.

Actinomycetales↗
PubMed1995

Solution structure of a cellulose-binding domain from Cellulomonas fimi by nuclear magnetic resonance spectroscopy.

Multidimensional, multinuclear nuclear magnetic resonance spectroscopy combined with dynamical simulated annealing has been used to determine the structure of a 110 amino acid cellulose-binding domain (CBD) from Cex, a beta-1,4-glycanase from the bacterium Cellulomonas fimi (CBDcex). An experimental data set comprising 1795 interproton NOE-derived restraints, 50 phi, 34 chi 1, and 106 hydrogen bond restraints was used to calculate 20 final structures. The calculated structures have an average root-mean-square (rms) deviation about the mean structure of 0.41 A for backbone atoms and 0.67 A for all heavy atoms when fitted over the secondary structural elements. Chromatography, ultracentrifugation, and 15N NMR relaxation experiments demonstrate that CBDcex is a dimer in solution. While attempts to measure NOEs across the dimer interface were unsuccessful, a computational strategy was employed to generate dimer structures consistent with the derived data set. The results from the dimer calculations indicate that, while the monomer topologies produced in the context of the dimer can be variable, the relative positioning of secondary structural elements and side chains present in the monomer are restored upon dimer formation. CBDcex forms an extensive beta-sheet structure with a beta-barrel fold. Titration with cellohexaose, [beta-D-glucopyranosyl-(1,4)]5-D-glucose, establishes that Trp 54 and 72 participate in cellulose binding. Analysis of the structure shows that these residues are adjacent in space and exposed to solvent. Together with other proximate hydrophilic residues, these residues form a carbohydrate-binding cleft, which appears to be a feature common to all CBDs of the same family.

Actinomycetales↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1994

C1-Cx revisited: intramolecular synergism in a cellulase.

Endoglucanase A (CenA) from the bacterium Cellulomonas fimi is composed of a catalytic domain and a nonhydrolytic cellulose-binding domain that can function independently. The individual domains interact synergistically in the disruption and hydrolysis of cellulose fibers. This intramolecular synergism is distinct from the well-known intermolecular synergism between individual cellulases. The catalytic domain corresponds to the hydrolytic Cx system and the cellulose-binding domain corresponds to the nonhydrolytic C1 system postulated by Reese et al. [Reese, E. T., Sui, R. G. H. & Levinson, H. S. (1950) J. Bacteriol. 59, 485-497] to be required for the hydrolysis of cellulose.

Actinomycetales↗
PubMed1994

Crystal structure of the catalytic domain of the beta-1,4-glycanase cex from Cellulomonas fimi.

beta-1,4-Glycanases, principally cellulases and xylanases, are responsible for the hydrolysis of plant biomass. The bifunctional beta-1,4-xylanase/glucanase Cex from the bacterium Cellulomonas fimi, one of a large family of cellulases/xylanases, depolymerizes oligosaccharides and releases a disaccharide unit from the substrate nonreducing end. Hydrolysis occurs with net retention of the anomeric configuration of the sugar through a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate. The active site nucleophile, Glu233, has been unambiguously identified by trapping of such an intermediate [Tull et al. (1991) J. Biol. Chem. 266, 15621-15625] and the acid/base catalyst, Glu127, by detailed kinetic analysis of mutants [MacLeod et al. (1994) Biochemistry 33, 6371-6376]. However, little is known about the enzyme's overall folding and its active site architecture. We report here the high-resolution crystal structure of the catalytic domain of Cex. The atomic structure refinement results in a model that includes 2400 protein atoms and 45 water molecules, with an R-factor of 0.217 for data extending to 1.8-A resolution. The protein forms an eight-parallel-stranded alpha/beta-barrel, which is a novel folding pattern for a microbial beta-glycanase. The active site, inferred from the location of Glu233, Glu127, and other conserved residues, is an open cleft on the carboxy-terminal end of the alpha/beta-barrel. An extensive hydrogen-bonding network stabilizes the ionization states of the key residues; in particular, the Asp235-His205-Glu233 hydrogen-bonding network may play a role in modulating the ionization state of Glu233 and in controlling local charge balance during the reaction.

Binding Sites↗
PubMed1994

Changes in the molecular-size distribution of insoluble celluloses by the action of recombinant Cellulomonas fimi cellulases.

Specific patterns of attacks of cotton, bacterial cellulose and bacterial microcrystalline cellulose (BMCC) by recombinant cellulases of Cellulomonas fimi were investigated. Molecular-size distributions of the celluloses were determined by high-performance size-exclusion chromatography. Chromatography of cotton and bacterial celluloses revealed single major peaks centered over progressively lower molecular-mass positions during attack by endoglucanase CenA. In advanced stages, a second peak appeared at very low average size (approx. 11 glucosyl units); ultimate weight losses were approximately 30%. The isolated catalytic domain of CenA, p30, gave results very similar to those with complete CenA. CenA did not effectively depolymerize or solubilize BMCC significantly. Molecular-size distributions of cotton and bacterial cellulose incubated with endoglucanases CenB or CenD exhibited one major peak regardless of incubation time; low-molecular-mass fragments did not accumulate. Weight losses were 40 and 35% respectively. The single peak shifted to lower-molecular-mass positions as incubation continued, but high-molecular-mass material persisted. CenB and CenD readily attacked and solubilized BMCC (approx. 70%). We conclude that CenA attacks cellulose by preferentially cleaving completely through the cellulose microfibrils at the amorphous sites, and much more slowly by degrading the crystalline surfaces. Conversely, CenB and CenD cleave the amorphous regions much less efficiently while vigorously degrading the surfaces of the crystalline regions of the microfibrils.

Cellulase↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1994

A streptavidin-cellulose-binding domain fusion protein that binds biotinylated proteins to cellulose.

A fusion protein, Sta-CBDCex, which comprises streptavidin with a cellulose-binding domain (CBDCex) fused to its C terminus, was produced in the cytoplasm of Escherichia coli, where it formed inclusion bodies. Renatured Sta-CBDCex, recovered from the inclusion bodies, adsorbed to Avicel, a microcrystalline cellulose. The cellulose-bound Sta-CBDCex in turn bound biotinylated alkaline phosphatase or biotinylated beta-glucosidase. The immobilized beta-glucosidase remained fully active during 2 weeks of continuous column operation at 50 degrees C.

Biotin↗
PubMed1994

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.

Adsorption↗