The oligosaccharide-producing multifunctional amylase-N (OPMA-N) is a novel multifunctional amylase and exhibits both hydrolytic and transglycosyl activities, but the molecular mechanism for its multiple catalytic activities is still unknown. Our research investigates the possible catalytic roles of a Trp residue in OPMA-N (Trp358) which is not only near the catalytic site Glu356 but also highly conserved in glycoside hydrolase subfamily 20 (the neopullulanase subfamily). Site-directed mutageneses at this site reveal that the size and charge of the occupying amino acid directly affect substrate binding, orientation of other crucial catalytic residues, the catalytic specificity, the oligomer formation, as well as the thermal stability of the enzyme. These findings may be useful in elucidating the different mechanisms of the multiple catalytic activities of multifunctional amylase OPMA-N and hence for developing an improved multifunctional amylase for the preparation of isomaltooligosaccharides.