hymecromone and 4-methylumbelliferylcellobioside

Endocellulase E2 from the thermophilic bacterium Thermomonospora fusca is a member of glycosyl-hydrolase family 6 and is active from pH 4 to 10. Enzymes in this family hydrolyze beta-1,4-glycosidic bonds with inversion of the stereochemistry at the anomeric carbon. The X-ray crystal structures of two family 6 enzymes have been determined, and four conserved aspartic acid residues are found in or near the active sites of both. These residues have been mutated in another family 6 enzyme, Cellulomonas fimi CenA, and evidence was found for both a catalytic acid and a catalytic base. The corresponding residues in E2 (D79, D117, D156, and D265) were mutated, and the mutant genes were expressed in Streptomyces lividans. The mutant enzymes were purified and assayed for activity on three cellulosic substrates and 2, 4-dinitrophenyl-beta-D-cellobioside. Activity on phosphoric acid-swollen cellulose was measured as a function of pH for selected mutant enzymes. Binding affinities for each mutant enzyme were measured for two fluorescent ligands and cellotriose, and circular dichroism spectra were recorded. The results show that the roles of D117 and D156 are the same as those for the corresponding residues in CenA; D117 is the catalytic acid, and D156 raises the pK(a) of D117. No specific function was assigned to the CenA residue corresponding to D79, but in E2, this residue also assists in raising the pK(a) of D117 and is important for catalytic activity. The D265N mutant retained 7% of the wild-type activity, indicating that this residue is not playing the role of the catalytic base. Experiments were conducted to rule out contamination of the D265 enzymes by either wild-type E2 or an endogenous S. lividans CMCase.

Topics: Actinomycetales; Binding Sites; Carboxymethylcellulose Sodium; Cellobiose; Cellulase; Circular Dichroism; Deuterium Oxide; Filtration; Glucosides; Hydrogen-Ion Concentration; Hymecromone; Mutagenesis, Site-Directed; Paper; Phosphoric Acids; Solvents; Trisaccharides

The determination of the high-resolution structure of the Thermomonospora fusca endocellulase E2 catalytic domain makes it ideal for exploring cellulase structure-function relationships. Here we present binding parameters (Kd, DeltaH degrees, and DeltaS degrees) describing the interaction of E2 with 4-methylumbelliferyl glycosides, determined by titrating the quenching of ligand fluorescence in equilibrium binding experiments. Quenched MU(Glc)2/E2 complexes were used as indicators in displacement titrations to measure the binding of natural glycosides and also of a nonhydrolyzable cellotetraose analogue. Binding of MU(Glc)2 and cellotriose were also determined by titration calorimetry. The results show that E2 binds glycosides exclusively in its active-site cleft, with high affinity and specificity. The observed patterns of ligand hydrolysis and the results with MU(Glc)2 as a substrate indicated that ligands bound to E2 with their nonreducing ends in position -2, consistent with the position of cellobiose in the E2cd structure. Polymerase chain reaction (PCR) mutagenesis of the conserved residue Tyr 73 (in E2 binding subsite -1) to Phe and Ser produced enzymes with lower activity but higher binding affinities, indicating that the volume of the subsite -1 binding pocket is crucial for enzyme function. Similarly, MUXylGlc (with its xylosyl unit located in position -1) bound with 100-fold higher affinity than MU(Glc)2. These results are similar to those for the related Trichoderma reesei exocellulase CBH II. The binding data were compared with that previously reported for CBH II and interpreted in terms of the functional differences between endo- and exocellulases.

Topics: Actinomycetales; Bacterial Proteins; Binding Sites; Calorimetry; Cellobiose; Cellulase; Glucose; Glucosides; Glycosides; Hydrogen-Ion Concentration; Hymecromone; Kinetics; Ligands; Mutagenesis, Site-Directed; Phenylalanine; Serine; Spectrometry, Fluorescence; Thermodynamics; Titrimetry; Tyrosine

The gram-negative bacterium Myxobacter sp. AL-1 produces chitosanase-cellulase activity that is maximally excreted during the stationary phase of growth. Carboxymethylcellulase zymogram analysis revealed that the enzymatic activity was correlated with two bands of 32 and 35 kDa. Ion-exchange-chromatography-enriched preparations of the 32-kDa enzyme were capable of degrading the cellulose fluorescent derivatives 4-methylumbelliferyl-beta-D-cellobioside and 4-methylumbelliferyl-beta-D-cellotrioside. These enzymatic preparations also showed a greater capacity at 70 degrees C than at 42 degrees C to degrade chitosan oligomers of a minimum size of six units. Conversely, the beta-1,4 glucanolytic activity was more efficient at attacking carboxymethylcellulose and methylumbelliferyl-cellotrioside at 42 degrees C than at 70 degrees C. The 32-kDa enzyme was purified more than 800-fold to apparent homogeneity by a combination of ion-exchange and molecular-exclusion chromatography. Amino-terminal sequencing indicated that mature chitosanase-cellulase shares more than 70% identity with endocellulases produced by strains DLG, PAP115, and 168 of the gram-positive microorganism Bacillus subtilis.

Topics: Amino Acid Sequence; Bacillus subtilis; Carboxymethylcellulose Sodium; Cellobiose; Cellulase; Cellulose; Chitin; Chitosan; Chromatography, Ion Exchange; Glycoside Hydrolases; Hymecromone; Molecular Sequence Data; Myxococcales; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Trisaccharides

Using 4-methylumbelliferyl (MUF) beta-D-cellobioside as a substrate, the ability of cellobiohydrolase I from Trichoderma longibrachiatum to catalyze transglycosylation has been demonstrated. At substrate concentrations greater than 2 mM, the formation of MUF-tetrasaccharide was detected using HPLC. In the course of enzymatic reaction, a concentration of the transglycosylation product passed through a maximum, since at later stages of the reaction the product was further hydrolyzed. At MUF-beta-D-cellobioside concentrations of 2-10 mM, the maximum weight content of MUF-tetrasaccharide amounted to 1-4% of the total content of saccharides. In the reaction system, containing 2.5 mM MUF-beta-D-cellobioside and 10 mM MUF-beta-D-glucoside, MUF-trisaccharide was formed as the main transglycosylation product. In hydrolysis of natural substrates (cellulose and cellotriose) in the presence of MUF-beta-D-glucoside a formation of MUF-trisaccharide was also observed.

Topics: Cellobiose; Cellulose; Cellulose 1,4-beta-Cellobiosidase; Chromatography, High Pressure Liquid; Glucosides; Glycoside Hydrolases; Glycosylation; Hymecromone; Models, Chemical; Trichoderma; Trisaccharides