@article {4856,
	title = {Binding site analysis of cellulose binding domain CBDN1 from endoglucanse C of Cellulomonas fimi by site-directed mutagenesis},
	journal = {Biochemistry},
	volume = {39},
	number = {30},
	year = {2000},
	note = {ISI Document Delivery No.: 339RJTimes Cited: 17Cited Reference Count: 59},
	month = {Aug},
	pages = {8844-8852},
	type = {Article},
	abstract = {Endoglucanase C (CenC), a beta 1,4 glucanase from the soil bacterium Cellulomonas fimi, binds to amorphous cellulose via two homologous cellulose binding domains, termed CBDN1 and CBDN2. in 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 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 CBDN1 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.},
	keywords = {4-GLUCANASE CENC, BETA-1, ESCHERICHIA-COLI, LIGAND-BINDING, LIMITED PROTEOLYSIS, MECHANISM, N-ACETYLGLUCOSAMINE, NUCLEAR-MAGNETIC-RESONANCE, REESEI CELLOBIOHYDROLASE-I, TRICHODERMA-REESEI, TRYPTOPHAN RESIDUES},
	isbn = {0006-2960},
	url = {<Go to ISI>://000088491500018},
	author = {Kormos, J. and Johnson, P. E. and Brun, E. and Tomme, P. and McIntosh, L. P. and Haynes, C. A. and Kilburn, D. G.}
}
@article {4842,
	title = {Hydrogen bonding and catalysis: A novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase},
	journal = {Journal of Molecular Biology},
	volume = {299},
	number = {1},
	year = {2000},
	note = {ISI Document Delivery No.: 318NCTimes Cited: 96Cited Reference Count: 58},
	month = {May},
	pages = {255-279},
	type = {Article},
	abstract = {The pH optima of family 11 xylanases are well correlated with the nature of the residue adjacent to the acid/base catalyst. In xylanases that function optimally under acidic conditions, this residue is aspartic acid, whereas it is asparagine in those that function under more alkaline conditions. Previous studies of wild-type (WT) Bacillus circulans xylanase (BCX), with an asparagine residue at position 35, demonstrated that its pH-dependent activity follows the ionization states of the nucleophile Glu78 (pK(a) 4.6) and the acid/base catalyst Glu172 (pK(a) 6.7). As predicted from sequence comparisons, substitution of this asparagine residue with an aspartic acid residue (N35D BCX) shifts its pH optimum from 5.7 to 4.6, with an similar to 20 \% increase in activity. The bell-shaped pH-activity profile of this mutant enzyme follows apparent pK(a) values of 3.5 and 5.8. Based on C-13-NMR titrations, the predominant pK(a) values of its active-site carboxyl groups are 3.7 (Asp35), 5.7 (Glu78) and 8.4 (Glu172). Thus, in contrast to the WT enzyme, the pH-activity profile of N35D BCX appears to be set by Asp35 and Glu78. Mutational, kinetic, and structural studies of N35D BCX, both in its native and covalently modified 2-fluoro-xylobiosyl glycosyl-enzyme intermediate states, reveal that the xylanase still follows a double-displacement mechanism with Glu78 serving as the nucleophile. We therefore propose that Asp35 and Glu172 function together as the general acid/base catalyst, and that N35D BCX exhibits a "reverse protonation" mechanism in which it is catalytically active when Asp35, with the lower pK(a), is protonated, while Glu78, with the higher pK(a), is deprotonated. This implies that the mutant enzyme must have an inherent catalytic efficiency at least 100-fold higher than that of the parental WT, because only similar to 1\% of its population is in the correct ionization state for catalysis at its pH optimum. The increased efficiency of N35D BCX, and by inference all "acidic" family 11 xylanases, is attributed to the formation of a short (2.7 Angstrom) hydrogen bond between Asp35 and Glu172, observed in the crystal structure of the glycosyl-enzyme intermediate of this enzyme, that will substantially stabilize the transition state for glycosyl transfer. Such a mechanism may be much more commonly employed than is generally realized, necessitating careful analysis of the pH-dependence of enzymatic catalysis. (C) 2000 Academic Press.},
	keywords = {3-DIMENSIONAL STRUCTURE, 4-BETA-XYLANASES, ACTIVE-SITE, BACILLUS-CIRCULANS XYLANASE, CYCLODEXTRIN GLYCOSYLTRANSFERASE, electrostatics, ENDO-1, GLYCOSYL-ENZYME INTERMEDIATE, ISOTOPE, MOLECULAR-WEIGHT, NMR, NUCLEAR-MAGNETIC-RESONANCE, nucleophile, PANCREATIC ALPHA-AMYLASE, pH-dependent enzyme mechanism, SHIFT, TRICHODERMA-REESEI, X-RAY CRYSTALLOGRAPHY, X-ray structure},
	isbn = {0022-2836},
	url = {<Go to ISI>://000087289400019},
	author = {Joshi, M. D. and Sidhu, G. and Pot, I. and Brayer, G. D. and Withers, S. G. and McIntosh, L. P.}
}
@article {4667,
	title = {Sugar ring distortion in the glycosyl-enzyme intermediate of a family G/11 xylanase},
	journal = {Biochemistry},
	volume = {38},
	number = {17},
	year = {1999},
	note = {ISI Document Delivery No.: 193YMTimes Cited: 122Cited Reference Count: 50},
	month = {Apr},
	pages = {5346-5354},
	type = {Article},
	abstract = {The 1.8 Angstrom resolution structure of the glycosyl-enzyme intermediate formed on the retaining beta-1,4-xylanase from Bacillus circulans has been determined using X-ray crystallographic techniques. The 2-fluoro-xylose residue bound in the -1 subsite adopts a B-2,B-5 (boat) conformation, allowing atoms C5, O5, C1, and C2 of the sugar to achieve coplanarity as required at the oxocarbenium ion-like transition states of the double-displacement catalytic mechanism. Comparison of this structure to that of a mutant of this same enzyme noncovalently complexed with xylotetraose [Wakarchuk et al. (1994) Protein Sci. 3, 467-475] reveals a number of differences beyond the distortion of the sugar moiety. Most notably, a bifurcated hydrogen bond interaction is formed in the glycosyl-enzyme intermediate involving H-eta of Tyr69, the endocyclic oxygen (O5) of the xylose residue in the -1 subsite, and O-epsilon 2 of the catalytic nucleophile, Glu78. To gain additional understanding of the role of Tyr69 at the active site of this enzyme, we also determined the 1.5 Angstrom resolution structure of the catalytically inactive Tyr69Phe mutant. Interestingly, no significant structural perturbation due to the loss of the phenolic group is observed. These results suggest that the interactions involving the phenolic group of Tyr69, O5 of the proximal saccharide, and Glu78 O-epsilon 2 important for the catalytic mechanism of this enzyme, and it is proposed that, through charge redistribution, these interactions serve to stabilize the oxocarbenium-like ion of the transition state. Studies of the covalent glycosyl-enzyme intermediate of this xylanase also provide insight into specificity, as contacts with C5 of the xylose moiety exclude sugars with hydroxymethyl substituents, and the mechanism of catalysis, including aspects of stereoelectronic theory as applied to glycoside hydrolysis.},
	keywords = {ACTIVE-SITE RESIDUES, BACILLUS-CIRCULANS XYLANASE, beta-glucosidase, CELLULOMONAS-FIMI, glycosidase, IDENTIFICATION, LEAVING GROUP, MECHANISM, REACTION COORDINATE, TRICHODERMA-REESEI},
	isbn = {0006-2960},
	url = {<Go to ISI>://000080165400009},
	author = {Sidhu, G. and Withers, S. G. and Nguyen, N. T. and McIntosh, L. P. and Ziser, L. and Brayer, G. D.}
}
@article {4235,
	title = {Characterization of a buried neutral histidine in Bacillus circulans xylanase: Internal dynamics and interaction with a bound water molecule},
	journal = {Biochemistry},
	volume = {37},
	number = {7},
	year = {1998},
	note = {ISI Document Delivery No.: YZ886Times Cited: 13Cited Reference Count: 59},
	month = {Feb},
	pages = {1810-1818},
	type = {Article},
	abstract = {NMR spectroscopy was used to characterize the dynamic behavior of His149 in Bacillus circulans xylanase (BCX) and its interaction with an internal water molecule. Rate constants for the specific acid-and base-catalyzed exchange following bimolecular kinetics (EX2) of the nitrogen-bonded H-epsilon 2 of this buried, neutral histidine were determined, At pD(min) 7.0 and 30 degrees C, the lifetime for this proton is 9.9 h, corresponding to a protection factor of similar to 10(7) relative to that predicted for an exposed histidine, The apparent activation energies measured for specific acid and base catalysis (7.0 and 17.4 kcal/mol) indicate that exchange occurs via local structural fluctuations. Consistent with its buried environment, the N-epsilon 2-H bond vector of His149 shows restricted mobility, as evidenced by an order parameter S-2 = 0.83 determined from N-15 relaxation measurements, The crystal structure of BCX reveals that a conserved, buried water hydrogen-bonds to the H-epsilon 2 of His149. Strong support for this interaction in solution is provided by the observation of a negative nuclear Overhauser effect (NOE) and positive rotating-frame Overhauser effect (ROE) between His149 H-epsilon 2 and a water molecule with the same chemical shift as the bulk solvent. However, the chemical shift of H-epsilon 2 (12.2 ppm) and a D/H fractionation factor close to unity (0.89 +/- 0.02) indicate that this is nor a so-called low-barrier hydrogen bond. Lower and upper bounds on the lifetime of the internal water are estimated to be 10(-8) and 10(-3) s. Therefore the chemical exchange of solvent protons with those of His149 H-epsilon 2 and the diffusion or physical exchange of the internal water to which the histidine is hydrogen-bonded differ in rate by over 7 orders of magnitude.},
	keywords = {ACTIVE-SITE, AMIDE PROTON-EXCHANGE, ASSIGNMENTS, DOMAIN, HYDROGEN-BOND, NMR-SPECTROSCOPY, NUCLEAR MAGNETIC-RESONANCE, PROTEINS, relaxation, TRICHODERMA-REESEI},
	isbn = {0006-2960},
	url = {<Go to ISI>://000072304200010},
	author = {Connelly, G. P. and McIntosh, L. P.}
}
@article {3708,
	title = {Interaction of soluble cellooligosaccharides with the N-terminal cellulose-binding domain of Cellulomonas fimi CenC .2. NMR and ultraviolet absorption spectroscopy},
	journal = {Biochemistry},
	volume = {35},
	number = {44},
	year = {1996},
	note = {ISI Document Delivery No.: VR098Times Cited: 68Cited Reference Count: 46},
	month = {Nov},
	pages = {13895-13906},
	type = {Article},
	abstract = {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 H-1 and N-15 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 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.},
	keywords = {BACKBONE H-1, CHEMICAL-SHIFTS, ESCHERICHIA-COLI, EXPRESSION, HIGH-LEVEL, NUCLEAR-MAGNETIC-RESONANCE, PROTEIN, REESEI CELLOBIOHYDROLASE-I, SEQUENCE, TRICHODERMA-REESEI, TRYPTOPHAN RESIDUES},
	isbn = {0006-2960},
	url = {<Go to ISI>://A1996VR09800005},
	author = {Johnson, P. E. and Tomme, P. and Joshi, M. D. and McIntosh, L. P.}
}
@article {3621,
	title = {Probing the role of tryptophan residues in a cellulose-binding domain by chemical modification},
	journal = {Protein Science},
	volume = {5},
	number = {11},
	year = {1996},
	note = {ISI Document Delivery No.: VQ786Times Cited: 41Cited Reference Count: 34},
	month = {Nov},
	pages = {2311-2318},
	type = {Article},
	abstract = {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 p-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.},
	keywords = {absorbance spectroscopy, ACTIVE-SITE, CELLOBIOHYDROLASE-I, cellulose-binding, ENDOGLUCANASE, ESCHERICHIA-COLI, FIMI, fluorescence spectroscopy, N-bromosuccinimide, NMR, NUCLEAR-MAGNETIC-RESONANCE, SCHIZOPHYLLUM-COMMUNE, SPECTROSCOPY, TRICHODERMA-REESEI, XYLANASE-A},
	isbn = {0961-8368},
	url = {<Go to ISI>://A1996VQ78600017},
	author = {Bray, M. R. and Johnson, P. E. and Gilkes, N. R. and McIntosh, L. P. and Kilburn, D. G. and Warren, R. A. J.}
}
@article {3707,
	title = {Structure of the N-terminal cellulose-binding domain of Cellulomonas fimi CenC determined by nuclear magnetic resonance spectroscopy},
	journal = {Biochemistry},
	volume = {35},
	number = {45},
	year = {1996},
	note = {ISI Document Delivery No.: VT081Times Cited: 94Cited Reference Count: 90},
	month = {Nov},
	pages = {14381-14394},
	type = {Article},
	abstract = {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 Angstrom for all backbone atoms excluding disordered termini and 0.44 +/- 0.05 Angstrom 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.},
	keywords = {3-DIMENSIONAL SOLUTION STRUCTURE, C-13-ENRICHED PROTEINS, CHEMICAL-SHIFT, DISTANCE GEOMETRY, INDEX, ISOTOPICALLY-ENRICHED PROTEINS, LARGER PROTEINS, NMR-SPECTROSCOPY, REESEI CELLOBIOHYDROLASE-I, SIDE-CHAIN RESONANCES, TRICHODERMA-REESEI},
	isbn = {0006-2960},
	url = {<Go to ISI>://A1996VT08100039},
	author = {Johnson, P. E. and Joshi, M. D. and Tomme, P. and Kilburn, D. G. and McIntosh, L. P.}
}
