Molecular dynamic analysis of mutant Y195I alpha-cyclodextrin glycosyltransferase with switched product specificity from alpha-cyclodextrin to gamma-cyclodextrin

摘要

Alpha-cyclodextrin (alpha-CD) glycosyltransferase (alpha-CGTase) can convert starch into alpha-CD blended with various proportions of beta-cyclodextrin (beta-CD) and/or gamma-cyclodextrin (gamma-CD). In this study, we verified the catalytic characteristics of purified Y195I alpha-CGTase and elucidated the mechanism of action with molecular dynamic (MD) simulations. We found that purified Y195I alpha-CGTase produced less alpha-CD, slightly more beta-CD, and significantly more gamma-CD than wild-type alpha-CGTase. Correspondingly, alpha-CD-based K-m values increased, and beta-CD- and gamma-CD-based K-m values decreased. MD simulation studies revealed that the dynamic trajectories of the substrate oligosaccharide chain in the mutant CGTase binding site were significantly different from those in the wild-type enzyme, with reduced hydrophobic interaction, finally resulting in different product specificity and more gamma-CD formation.