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Douglas G. Kilburn - One of the best experts on this subject based on the ideXlab platform.
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glycosylation by pichia pastoris decreases the affinity of a family 2a carbohydrate binding module from Cellulomonas fimi a functional and mutational analysis
Biochemical Journal, 2001Co-Authors: Alisdair B Boraston, Antony R. J. Warren, Douglas G. KilburnAbstract:When produced by Pichia pastoris, three of the five Asn-Xaa-Ser/Thr sequences (corresponding to Asn-24, Asn-73 and Asn-87) in the carbohydrate-binding module CBM2a of xylanase 10A from Cellulomonas fimi are glycosylated. The glycans are of the high-mannose type, ranging in size from GlcNAc(2)Man(8) to GlcNAc(2)Man(14). The N-linked glycans block the binding of CBM2a to cellulose. Analysis of mutants of CBM2a shows that glycans on Asn-24 decrease the association constant (K(a)) for the binding of CBM2a to bacterial microcrystalline cellulose approx. 10-fold, whereas glycans on Asn-87 destroy binding. The K(a) of a mutant of CBM2a lacking all three N-linked glycosylation sites is the same when the polypeptide is produced by either Escherichia coli or P. pastoris and is approx. half that of wild-type CBM2a produced by E. coli.
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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
Protein Engineering, 2000Co-Authors: Bradley W Mclean, N R Gilkes, Charles A Haynes, Alisdair B Boraston, Mark R Bray, Douglas G. KilburnAbstract: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.
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binding site analysis of cellulose binding domain cbdn1 from endoglucanse c of Cellulomonas fimi by site directed mutagenesis
Biochemistry, 2000Co-Authors: Jeff Kormos, P Tomme, Charles A Haynes, Lawrence P Mcintosh, Philip E Johnson, Emmanual Brun, Douglas G. KilburnAbstract:Endoglucanase C (CenC), a ‚1,4 glucanase from the soil bacterium Cellulomonas fimi, binds to amorphous cellulose via two homologous cellulose binding domains, termed CBDN1 and CBDN2 .I n this work, the contributions of 10 amino acids within the binding cleft of CBDN1 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 ‚-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 CBDN1 as their replacement by alanine results in affinity decrements of approximately 100-fold on PASA, barley ‚-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.
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mannanase man26a from Cellulomonas fimi has a mannan binding module
Fems Microbiology Letters, 2000Co-Authors: Dominik Stoll, Douglas G. Kilburn, Henrik Stålbrand, Alisdair B Boraston, Bradley W Mclean, Anthony R J WarrenAbstract:A modular mannanase (Man26A) from the bacterium Cellulomonas fimi contains a mannan-binding module (Man26Abm) that binds to soluble but not to insoluble mannans. Man26Abm does not bind to cellulose, chitin or xylan. The Kd for binding of Man26Abm to locust bean gum (LBG) is ∼0.2 μM. Man26A is the first mannanase reported to contain a mannan-binding module.
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Analysis of Molecular Size Distributions of Cellulose Molecules during Hydrolysis of Cellulose by Recombinant Cellulomonas fimi β-1,4-Glucanases
Applied and Environmental Microbiology, 1998Co-Authors: Henrik Stålbrand, Douglas G. Kilburn, R. Antony J. Warren, Shawn D. Mansfield, John N. Saddler, Neil R. GilkesAbstract:Four β-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 β-1,4-glucan chains to enzyme attack. Endoglucanases and cellobiohydrolases were much more easily distinguished when the acid-swollen substrate was used.
N R Gilkes - One of the best experts on this subject based on the ideXlab platform.
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the effects of recombinant Cellulomonas fimi β 1 4 glycanases on softwood kraft pulp fibre and paper properties
Progress in Biotechnology, 2002Co-Authors: Shawn D. Mansfield, N R Gilkes, Antony R. J. Warren, D G KilburnAbstract:Abstract Recombinant Cellulomonas fimi β-1,4-endoglucanases (Cel5A and Cel6A) and cellobiohydrolases (Cel6B and Cel48A) were assessed for their capacity to selectively modify the physical and optical properties of handsheets derived from a fully bleached, never-dried softwood pulp. The isolated binding domain (Cel6A CBD) and catalytic domain (Cel6A CD) were also evaluated. Treatment with endoglucanases, particularly Cel6A CD, caused substantial damage to the intrinsic fibre strength of the kraft furnish, and consequently compromised the physical handsheet properties. In contrast, treatment with Cel48A produced beneficial modifications, including improvements in handsheet tensile strength following mechanical refining and various degrees of wet pressing. Cel6B and Cel6A CBD had very limited effects on the fibre characteristics, and therefore did not alter the quality of the handsheets produced. An examination of carbohydrate solubilization and changes in the degree of polymerisation of the polysaccharides indicated that the capacity for Cel48A to beneficially modify pulp and paper characteristics seems to be related to its capacity to selectively degrade cellulose-hemicellulose linkages, and release and modify xylan moieties. Furthermore, this enzyme preparation demonstrated limited carbohydrate dissolution, indicating that the observed modifications were attained with very little yield loss. These results suggest that Cel48A, and enzymes with similar glycanase activity should be considered and further evaluated for their potential to treat pulp fibres to improve paper properties.
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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
Protein Engineering, 2000Co-Authors: Bradley W Mclean, N R Gilkes, Charles A Haynes, Alisdair B Boraston, Mark R Bray, Douglas G. KilburnAbstract: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.
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analysis of molecular size distributions of cellulose molecules during hydrolysis of cellulose by recombinant Cellulomonas fimi β 1 4 glucanases
Applied and Environmental Microbiology, 1998Co-Authors: Henrik Stålbrand, Shawn D. Mansfield, John N. Saddler, D G Kilburn, Antony R. J. Warren, N R GilkesAbstract: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.
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Cellobiohydrolase B, a second exo-cellobiohydrolase from the cellulolytic bacterium Cellulomonas fimi.
The Biochemical journal, 1995Co-Authors: H Shen, N R Gilkes, R C Miller, D G Kilburn, R A WarrenAbstract: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.
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solution structure of a cellulose binding domain from Cellulomonas fimi by nuclear magnetic resonance spectroscopy
Biochemistry, 1995Co-Authors: E Ong, N R Gilkes, Douglas G. Kilburn, D R Muhandiram, Marees Harrisbrandts, Jeremy P Carver, Lewis E Kay, Timothy S HarveyAbstract: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.
Stephen G. Withers - One of the best experts on this subject based on the ideXlab platform.
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Characterization of a thermostable endoglucanase from Cellulomonas fimi ATCC484
Biochemistry and Cell Biology, 2017Co-Authors: Hirak Saxena, Bryan Hsu, Marc De Asis, Mirko Zierke, Lyann Sim, Stephen G. Withers, Warren W. WakarchukAbstract:Bacteria in the genus Cellulomonas are well known as secretors of a variety of mesophilic carbohydrate degrading enzymes (e.g., cellulases and hemicellulases), active against plant cell wall polysaccharides. Recent proteomic analysis of the mesophilic bacterium Cellulomonas fimi ATCC484 revealed uncharacterized enzymes for the hydrolysis of plant cell wall biomass. Celf_1230 (CfCel6C), a secreted protein of Cellulomonas fimi ATCC484, is a novel member of the GH6 family of cellulases that could be successfully expressed in Escherichia coli. This enzyme displayed very little enzymatic/hydrolytic activity at 30 °C, but showed an optimal activity around 65 °C, and exhibited a thermal denaturation temperature of 74 °C. In addition, it also strongly bound to filter paper despite having no recognizable carbohydrate binding module. Our experiments show that CfCel6C is a thermostable endoglucanase with activity on a variety of β-glucans produced by an organism that struggles to grow above 30 °C.
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specificity fingerprinting of retaining β 1 4 glycanases in the Cellulomonas fimi secretome using two fluorescent mechanism based probes
ChemBioChem, 2007Co-Authors: Omid Hekmat, Youngwan Kim, Antony R. J. Warren, Christine Florizone, Lindsay D Eltis, Stephen G. WithersAbstract:Functional proteomics methods are crucial for activity- and mechanism-based investigation of enzymes in biological systems at a post-translational stage. Glycosidases have central roles in cellular metabolism and its regulation, and their dysfunction can have detrimental effects. These enzymes also play key roles in biomass conversion. A functional profiling methodology was developed for direct, fluorescence-based, in-gel analysis of retaining beta-glycosidases. Two spectrally nonoverlapping fluorescent, mechanism-based probes containing different recognition elements for retaining cellulases and xylanases were prepared. The specificity-based covalent labelling of retaining glycanases by the two probes was demonstrated in model enzyme mixtures. Using the two probes and mass spectrometry, the secretomes of the biomass-converting bacterium Cellulomonas fimi, under induction by different polyglycan growth substrates, were analysed to obtain a specificity profile of the C. fimi retaining beta-glycanases. This is a facile strategy for the analysis of glycosidases produced by biomass-degrading organisms.
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characterization of a β n acetylhexosaminidase and a β n acetylglucosaminidase β glucosidase from Cellulomonas fimi
FEBS Journal, 2006Co-Authors: Dominik Stoll, Karen Rupitz, R A J Warren, Christoph Mayer, David J Vocadlo, Melanie Mah, Stephen G. WithersAbstract:The Gram-positive soil bacterium Cellulomonas fimi is shown to produce at least two intracellular β-N-acetylglucosaminidases, a family 20 β-N-acetylhexosaminidase (Hex20), and a novel family 3-β-N-acetylglucosaminidase/β-glucosidase (Nag3), through screening of a genomic expression library, cloning of genes and analysis of their sequences. Nag3 exhibits broad substrate specificity for substituents at the C2 position of the glycone: kcat/Km values at 25 °C were 0.066 s−1·mm−1 and 0.076 s−1·mm−1 for 4′-nitrophenyl β-N-acetyl-d-glucosaminide and 4′-nitrophenyl β-d-glucoside, respectively. The first glycosidase with this broad specificity to be described, Nag3, suggests an interesting evolutionary link between β-N-acetylglucosaminidases and β-glucosidases of family 3. Reaction by a double-displacement mechanism was confirmed for Nag3 through the identification of a glycosyl–enzyme species trapped with the slow substrate 2′,4′-dinitrophenyl 2-deoxy-2-fluoro-β-d-glucopyranoside. Hex20 requires the acetamido group at C2 of the substrate, being unable to cleave β-glucosides, since its mechanism involves an oxazolinium ion intermediate. However, it is broad in its specificity for the d-glucosyl/d-galactosyl configuration of the glycone: Km and kcat values were 53 µm and 482.3 s−1 for 4′-nitrophenyl β-N-acetyl-d-glucosaminide and 66 µm and 129.1 s−1 for 4′-nitrophenyl β-N-acetyl-d-galactosaminide.
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glycosynthase based synthesis of xylo oligosaccharides using an engineered retaining xylanase from Cellulomonas fimi
Organic and Biomolecular Chemistry, 2006Co-Authors: Youngwan Kim, Omid Hekmat, Antony R. J. Warren, David T Fox, Terrence Kantner, Lawrence P Mcintosh, Stephen G. WithersAbstract:Glycosynthases are synthetic enzymes derived from retaining glycosidases in which the catalytic nucleophile has been replaced. The mutation allows irreversible glycosylation of sugar acceptors using glycosyl fluoride donors to afford oligosaccharides without any enzymatic hydrolysis. Glycosynthase technology has proven fruitful for the facile synthesis of useful oligosaccharides, therefore the expansion of the glycosynthase repertoire is of the utmost importance. Herein, we describe for the first time a glycosynthase, derived from a retaining xylanase, that synthesizes a range of xylo-oligosaccharides. The catalytic domain of the retaining endo-1,4-β-xylanase from Cellulomonas fimi (CFXcd) was successfully converted to the corresponding glycosynthase by mutation of the catalytic nucleophile to a glycine residue. The mutant enzyme (CFXcd-E235G) was found to catalyze the transfer of a xylobiosyl moiety from α-xylobiosyl fluoride to either p-nitrophenyl β-xylobioside or benzylthio β-xylobioside to afford oligosaccharides ranging in length from tetra- to dodecasaccharides. These products were purified by high performance liquid chromatography in greater than 60% combined yield. 1H and 13C NMR spectroscopic analyses of the isolated p-nitrophenyl xylotetraoside and p-nitrophenyl xylohexaoside revealed that CFXcd-E235G catalyzes both the regio- and stereo-selective synthesis of xylo-oligosaccharides containing, exclusively, β-(1 → 4) linkages.
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active site peptide fingerprinting of glycosidases in complex mixtures by mass spectrometry discovery of a novel retaining β 1 4 glycanase in Cellulomonas fimi
Journal of Biological Chemistry, 2005Co-Authors: Omid Hekmat, Spencer J. Williams, Youngwan Kim, Stephen G. WithersAbstract:Abstract New proteomics methods are required for targeting and identification of subsets of a proteome in an activity-based fashion. Here, we report the first gel-free, mass spectrometry-based strategy for mechanism-based profiling of retaining β-endoglycosidases in complex proteomes. Using a biotinylated, cleavable 2-deoxy-2-fluoroxylobioside inactivator, we have isolated and identified the active-site peptides of target retaining β-1,4-glycanases in systems of increasing complexity: pure enzymes, artificial proteomes, and the secreted proteome of the aerobic mesophilic soil bacterium Cellulomonas fimi. The active-site peptide of a new C. fimi β-1,4-glycanase was identified in this manner, and the peptide sequence, which includes the catalytic nucleophile, is highly conserved among glycosidase family 10 members. The glycanase gene (GenBank™ accession number DQ146941) was cloned using inverse PCR techniques, and the protein was found to comprise a catalytic domain that shares ∼70% sequence identity with those of xylanases from Streptomyces sp. and a family 2b carbohydrate-binding module. The new glycanase hydrolyzes natural and artificial xylo-configured substrates more efficiently than their cello-configured counterparts. It has a pH dependence very similar to that of known C. fimi retaining glycanases.
R A J Warren - One of the best experts on this subject based on the ideXlab platform.
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characterization of a β n acetylhexosaminidase and a β n acetylglucosaminidase β glucosidase from Cellulomonas fimi
FEBS Journal, 2006Co-Authors: Dominik Stoll, Karen Rupitz, R A J Warren, Christoph Mayer, David J Vocadlo, Melanie Mah, Stephen G. WithersAbstract:The Gram-positive soil bacterium Cellulomonas fimi is shown to produce at least two intracellular β-N-acetylglucosaminidases, a family 20 β-N-acetylhexosaminidase (Hex20), and a novel family 3-β-N-acetylglucosaminidase/β-glucosidase (Nag3), through screening of a genomic expression library, cloning of genes and analysis of their sequences. Nag3 exhibits broad substrate specificity for substituents at the C2 position of the glycone: kcat/Km values at 25 °C were 0.066 s−1·mm−1 and 0.076 s−1·mm−1 for 4′-nitrophenyl β-N-acetyl-d-glucosaminide and 4′-nitrophenyl β-d-glucoside, respectively. The first glycosidase with this broad specificity to be described, Nag3, suggests an interesting evolutionary link between β-N-acetylglucosaminidases and β-glucosidases of family 3. Reaction by a double-displacement mechanism was confirmed for Nag3 through the identification of a glycosyl–enzyme species trapped with the slow substrate 2′,4′-dinitrophenyl 2-deoxy-2-fluoro-β-d-glucopyranoside. Hex20 requires the acetamido group at C2 of the substrate, being unable to cleave β-glucosides, since its mechanism involves an oxazolinium ion intermediate. However, it is broad in its specificity for the d-glucosyl/d-galactosyl configuration of the glycone: Km and kcat values were 53 µm and 482.3 s−1 for 4′-nitrophenyl β-N-acetyl-d-glucosaminide and 66 µm and 129.1 s−1 for 4′-nitrophenyl β-N-acetyl-d-galactosaminide.
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exploring the cellulose xylan specificity of the beta 1 4 glycanase cex from Cellulomonas fimi through crystallography and mutation
Biochemistry, 1998Co-Authors: V Notenboom, Stephen G. Withers, R A J Warren, C Birsan, David R. RoseAbstract:The retaining β-1,4-glycanase Cex from Cellulomonas fimi, a family 10 glycosyl hydrolase, hydrolyzes xylan 40-fold more efficiently than cellulose. To gain insight into the nature of its preference...
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mechanistic consequences of mutation of active site carboxylates in a retaining beta 1 4 glycanase from Cellulomonas fimi
Biochemistry, 1996Co-Authors: A M Macleod, Dedreia Tull, Karen Rupitz, R A J Warren, Stephen G. WithersAbstract:The exoglucanase/xylanase Cex from Cellulomonas fimi is a retaining glycosidase which functions via a two-step mechanism involving the formation and hydrolysis of a covalent glycosyl-enzyme interme...
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Degradation of cellulases in cultures of Cellulomonas fimi
FEMS Microbiology Letters, 1996Co-Authors: L.e. Sandercock, Neil R. Gilkes, Douglas G. Kilburn, A. Meinke, R A J WarrenAbstract:Endoglucanases CenA, CenB and CenD, cellobiohydrolases CbhA and CbhB, and the mixed function xylanase-exoglucanase Cex are degraded proteolytically in the supernatants of cultures of Cellulomonas fimi growing with cellulose. All of these polypeptides are modular. The initial sites of proteolysis are within or adjacent to the linkers connecting the modules, leading to the appearance of discrete fragments of the enzymes which retain the functions of the component modules.
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site directed mutation of the putative catalytic residues of endoglucanase cena from Cellulomonas fimi
Biochemistry, 1995Co-Authors: H G Damude, Stephen G. Withers, Douglas G. Kilburn, Robert C. Miller, R A J WarrenAbstract:The catalytic domains of beta-1,4-glucanases can be grouped into families of related amino acid sequences. The endoglucanase CenA from Cellulomonas fimi is a member of family B. All enzymes from this family are believed to hydrolyze beta-1,4-glucosidic bonds using a general acid-base catalytic mechanism resulting in inversion of anomeric configuration at the scissile bond. Three-dimensional structures for two cellulases from family B have been determined by X-ray crystallographic analysis. These structures show that there are four Asp residues which are in a position to function as acid catalyst, base catalyst, and/or transition state stabilizers. These aspartates are conserved in all members of family B. The roles of Asp216, Asp252, Asp287, and Asp392, the corresponding amino acids in CenA, were determined. These aspartates have been systematically replaced with alanine and glutamate via site-directed mutagenesis, and the resulting effect on activity, substrate specificity, and overall structure has been determined. Changes in overall structure were monitored using circular dichroism spectroscopy, and no significant differences between the wild-type and mutant proteins were found. Active site structure was also found to be intact as all proteins bound to a cellobiose affinity column. The Michaelis-Menten parameters of the enzyme were determined on 2,4-dinitrophenyl cellobioside as well as (carboxymethyl)-cellulose and phosphoric acid-swollen cellulose. Initial characterization of mutant proteins indicates that Asp252 and Asp392 are the acid and base catalysts, respectively, in CenA. Residue Asp287 appears to aid Asp252 in acid catalysis, and Asp216 is not absolutely required for catalysis.
Antony R. J. Warren - One of the best experts on this subject based on the ideXlab platform.
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specificity fingerprinting of retaining β 1 4 glycanases in the Cellulomonas fimi secretome using two fluorescent mechanism based probes
ChemBioChem, 2007Co-Authors: Omid Hekmat, Youngwan Kim, Antony R. J. Warren, Christine Florizone, Lindsay D Eltis, Stephen G. WithersAbstract:Functional proteomics methods are crucial for activity- and mechanism-based investigation of enzymes in biological systems at a post-translational stage. Glycosidases have central roles in cellular metabolism and its regulation, and their dysfunction can have detrimental effects. These enzymes also play key roles in biomass conversion. A functional profiling methodology was developed for direct, fluorescence-based, in-gel analysis of retaining beta-glycosidases. Two spectrally nonoverlapping fluorescent, mechanism-based probes containing different recognition elements for retaining cellulases and xylanases were prepared. The specificity-based covalent labelling of retaining glycanases by the two probes was demonstrated in model enzyme mixtures. Using the two probes and mass spectrometry, the secretomes of the biomass-converting bacterium Cellulomonas fimi, under induction by different polyglycan growth substrates, were analysed to obtain a specificity profile of the C. fimi retaining beta-glycanases. This is a facile strategy for the analysis of glycosidases produced by biomass-degrading organisms.
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glycosynthase based synthesis of xylo oligosaccharides using an engineered retaining xylanase from Cellulomonas fimi
Organic and Biomolecular Chemistry, 2006Co-Authors: Youngwan Kim, Omid Hekmat, Antony R. J. Warren, David T Fox, Terrence Kantner, Lawrence P Mcintosh, Stephen G. WithersAbstract:Glycosynthases are synthetic enzymes derived from retaining glycosidases in which the catalytic nucleophile has been replaced. The mutation allows irreversible glycosylation of sugar acceptors using glycosyl fluoride donors to afford oligosaccharides without any enzymatic hydrolysis. Glycosynthase technology has proven fruitful for the facile synthesis of useful oligosaccharides, therefore the expansion of the glycosynthase repertoire is of the utmost importance. Herein, we describe for the first time a glycosynthase, derived from a retaining xylanase, that synthesizes a range of xylo-oligosaccharides. The catalytic domain of the retaining endo-1,4-β-xylanase from Cellulomonas fimi (CFXcd) was successfully converted to the corresponding glycosynthase by mutation of the catalytic nucleophile to a glycine residue. The mutant enzyme (CFXcd-E235G) was found to catalyze the transfer of a xylobiosyl moiety from α-xylobiosyl fluoride to either p-nitrophenyl β-xylobioside or benzylthio β-xylobioside to afford oligosaccharides ranging in length from tetra- to dodecasaccharides. These products were purified by high performance liquid chromatography in greater than 60% combined yield. 1H and 13C NMR spectroscopic analyses of the isolated p-nitrophenyl xylotetraoside and p-nitrophenyl xylohexaoside revealed that CFXcd-E235G catalyzes both the regio- and stereo-selective synthesis of xylo-oligosaccharides containing, exclusively, β-(1 → 4) linkages.
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the effects of recombinant Cellulomonas fimi β 1 4 glycanases on softwood kraft pulp fibre and paper properties
Progress in Biotechnology, 2002Co-Authors: Shawn D. Mansfield, N R Gilkes, Antony R. J. Warren, D G KilburnAbstract:Abstract Recombinant Cellulomonas fimi β-1,4-endoglucanases (Cel5A and Cel6A) and cellobiohydrolases (Cel6B and Cel48A) were assessed for their capacity to selectively modify the physical and optical properties of handsheets derived from a fully bleached, never-dried softwood pulp. The isolated binding domain (Cel6A CBD) and catalytic domain (Cel6A CD) were also evaluated. Treatment with endoglucanases, particularly Cel6A CD, caused substantial damage to the intrinsic fibre strength of the kraft furnish, and consequently compromised the physical handsheet properties. In contrast, treatment with Cel48A produced beneficial modifications, including improvements in handsheet tensile strength following mechanical refining and various degrees of wet pressing. Cel6B and Cel6A CBD had very limited effects on the fibre characteristics, and therefore did not alter the quality of the handsheets produced. An examination of carbohydrate solubilization and changes in the degree of polymerisation of the polysaccharides indicated that the capacity for Cel48A to beneficially modify pulp and paper characteristics seems to be related to its capacity to selectively degrade cellulose-hemicellulose linkages, and release and modify xylan moieties. Furthermore, this enzyme preparation demonstrated limited carbohydrate dissolution, indicating that the observed modifications were attained with very little yield loss. These results suggest that Cel48A, and enzymes with similar glycanase activity should be considered and further evaluated for their potential to treat pulp fibres to improve paper properties.
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glycosylation by pichia pastoris decreases the affinity of a family 2a carbohydrate binding module from Cellulomonas fimi a functional and mutational analysis
Biochemical Journal, 2001Co-Authors: Alisdair B Boraston, Antony R. J. Warren, Douglas G. KilburnAbstract:When produced by Pichia pastoris, three of the five Asn-Xaa-Ser/Thr sequences (corresponding to Asn-24, Asn-73 and Asn-87) in the carbohydrate-binding module CBM2a of xylanase 10A from Cellulomonas fimi are glycosylated. The glycans are of the high-mannose type, ranging in size from GlcNAc(2)Man(8) to GlcNAc(2)Man(14). The N-linked glycans block the binding of CBM2a to cellulose. Analysis of mutants of CBM2a shows that glycans on Asn-24 decrease the association constant (K(a)) for the binding of CBM2a to bacterial microcrystalline cellulose approx. 10-fold, whereas glycans on Asn-87 destroy binding. The K(a) of a mutant of CBM2a lacking all three N-linked glycosylation sites is the same when the polypeptide is produced by either Escherichia coli or P. pastoris and is approx. half that of wild-type CBM2a produced by E. coli.
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mannan degrading enzymes from Cellulomonas fimi
Applied and Environmental Microbiology, 1999Co-Authors: Dominik Stoll, Henrik Stålbrand, Antony R. J. WarrenAbstract:The genes man26a and man2A from Cellulomonas fimi encode mannanase 26A (Man26A) and β-mannosidase 2A (Man2A), respectively. Mature Man26A is a secreted, modular protein of 951 amino acids, comprising a catalytic module in family 26 of glycosyl hydrolases, an S-layer homology module, and two modules of unknown function. Exposure of Man26A produced by Escherichia coli to C. fimi protease generates active fragments of the enzyme that correspond to polypeptides with mannanase activity produced by C. fimi during growth on mannans, indicating that it may be the only mannanase produced by the organism. A significant fraction of the Man26A produced by C. fimi remains cell associated. Man2A is an intracellular enzyme comprising a catalytic module in a subfamily of family 2 of the glycosyl hydrolases that at present contains only mammalian β-mannosidases.