Active-site mutations which change the substrate specificity of the Clostridium stercorarium cellulase CelZ - Implications for synergism

CelZ from the cellulolytic thermophile Clostridium stercorarium has been de scribed as a 'monomeric' cellulase able to effect both the endoglucanolytic hydrolysis of internal glycosidic linkages and the exoglucanolytic degrada tion from the chain ends in a processive mode of action. The putative catal ytic residues of this family 9 cellulase, Asp84 and Glu447 located within t he N-terminal domain of the modular protein, were replaced by site-directed mutagenesis. A minimized CelZ derivative (CelZC') comprising the catalytic domain and the adjacent cellulose-binding domain (CBD) family IIIc domain C' was used as target for mutagenesis. Six mutant enzymes and the unmodifie d CelZC' protein were purified to homogeneity and compared with respect to thermoactivity, substrate specificity, product profile and synergism. CD st udies revealed that no major changes to the overall structure of the protei ns had occurred. Replacement of either one or both catalytic residues compl etely eliminated the ability of CelZ to attack insoluble Avicel preparation s indicative of the exo-activity, whereas the endo-activity measured via hy drolysis of CM-cellulose was retained upon substitution of the catalytic ba se Asp84. Thus, endo-active CelZ mutants defective in the exo-activity were available for co-operativity studies with the C, stercorarium exoglucanase CelY. Synergism was found to be dependent on the endo-activity of CelZ. Mu tants Asp84Gly and Asp84Glu were able to enhance the degradation of crystal line cellulose significantly, although no products could be released from t his substrate by individual action of the mutants.