Role of the glutamate 332 residue in the transglycosylation activity of Thermus maltogenic amylase

A sequence alignment shows that residue 332 is conserved as glutamate in ma ltogenic amylases (MAases) and in other related enzymes such as cyclodextri nase and neopullulanase, while the corresponding position is conserved as h istidine in cr-amylases. We analyzed the role of Glu332 in the hydrolysis a nd the transglycosylation activity of Thermus MAase (ThMA) by site-directed mutagenesis. Replacing Glu332 with histidine reduced transglycosylation ac tivity significantly, but enhanced hydrolysis activity on alpha-(1,3)-, alp ha-(1,4)-, and alpha-(1,6)-glycosidic bonds relative to the wild-type (WT) enzyme. The mutant Glu332Asp had catalytic properties similar to those of t he WT enzyme, but the mutant Glu332Gln resulted in significantly decreased transglycosylation activity. These results suggest that an acidic side chai n at position 332 of MAase plays an important role in the formation and acc umulation of transfer products by modulating the relative rates of hydrolys is and transglycosylation. From the structure, we propose that an acidic si de chain at position 332, which is located in a pocket, is involved in alig ning the acceptor molecule to compete with water molecules in the nucleophi lic attack of the glycosyl-enzyme intermediate.