SITE-DIRECTED MUTAGENESIS OF ACTIVE-SITE RESIDUES IN A CLASS-I ENDOCHITINASE FROM CHESTNUT SEEDS

Despite the intensive research on plant chitinases, largely bolstered by their antifungal properties, little is known at present about the s tructure-activity relationships of these enzymes. Here we report the i dentification of essential active site residues in endochitinase Ch3, a class I enzyme abundant in chestnut seeds. Knowledge-based protein m odeling as well as structural and sequence comparisons were performed to identify potential catalytic residues. Different mutated proteins w ere then generated by site-directed mutagenesis, expressed in Escheric hia coli, and characterized for their chitinolytic activity. Glu124 an d Glu146, the only carboxylic residues propel ly located into the acti ve site cleft to participate in catalysis, were both mutated to Gin an d Asp, Our results suggest that Glu124 functions as the general acid c atalyst whereas Glu146 is likely to act as a general base. Other mutat ions involving three highly conserved active site residues, Gln173, Th r175, and Asn254, also impaired the chitinolytic activity of Ch3, The effects of these variants on the fungus Trichoderma viride revealed th at catalysis is not necessary for antifungal activity. Similarly to it s homologous nonenzymatic polypeptides hevein and stinging nettle lect in, the N-terminal chitin-binding domain of Ch3 appears to interfere i tself with hyphal growth.