Abstract
We have previously proposed that α-amylases and their related enzymes including neopullulanase are to be called an α-amylase family owing to their common functional and structural characteristics. In these α-amylase family enzymes, neopullulanase has a very interesting enzymatic characteristic, that is, in its active center, neopullulanase acts on both α- (1→4) - and α- (1→6) -glucosidic linkages of polysaccharides, and even catalyzes both hydrolysis and transglycosylation reactions. Hence, using this enzyme, we expected to demonstrate a hypothesis that the enzymes belonging to α-amylase family can be altered each other by protein engineering. However, the 3-D structure of neopullulanase is still unknown. Here, the 3-D structure is predicted on the basis of the X-ray crystal structure of Taka-amylase A by homology modeling. Moreover, the enzyme-substrate structures which are difficult to be elucidated by experimental technique, are predicted by docking-study and molecular dynamics calculation. Based on these informations, we successfully altered the substrate specificity toward α-(1→6) -branched oligosaccharides and pullulan by manipulating the volume of side chain of 11e358, which was located in the active cleft. It is shown that I358W mutant was rather similar to a-amylase type enzyme that can not act on α- (1→6) -linkages. We also successfully altered the reaction speci-ficity toward transglycosylation by manipulating the hydrophobic environment near the entrance route of the water molecule which was probably used in hydrolysis process. It is shown that all the mutants which were substituted by more hydrophobic amino acids had high activities for transglycosylation.
