Mechanistic studies of active site mutants of Thermomonospora fusca endocellulase E2.
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.
Wolfgang
D E
DE
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.
Wilson
D B
DB
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002475:Cellobiose / Q000031:analogs & derivatives / Q000378:metabolism; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism*; D002942:Circular Dichroism; D017666:Deuterium Oxide; D005374:Filtration / Q000295:instrumentation; D005960:Glucosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010209:Paper; D010756:Phosphoric Acids / Q000378:metabolism; D012997:Solvents; D014312:Trisaccharides / Q000378:metabolism
C109060:4-methylumbelliferyl-beta-D-cellotrioside; D005960:Glucosides; D010756:Phosphoric Acids; D012997:Solvents; D014312:Trisaccharides; D002475:Cellobiose; C034170:4-nitrophenyl beta-cellobioside; D006923:Hymecromone; C014111:methylumbelliferyl-beta-D-xyloside; C023872:mannotriose; C068475:4-methylumbelliferylcellobioside; C030242:phosphoric acid; C056373:endoglucanase 2; D002480:Cellulase; D017666:Deuterium Oxide; D002266:Carboxymethylcellulose Sodium