Mechanistic insights into cyclodextrins as substrates and inhibitors of GH57 family amylopullulanase from Aquifex aeolicus

Abstract

Maltooligosaccharides (MOs) have gained significant attention in the food and pharmaceutical industries owing to their valuable functional properties, including controlled sweetness, digestibility, and enhanced bioavailability. However, conventional MOs is production involves complex processing steps and significant production costs. A potential high-efficiency synthesis of specific MOs can be achieved through the ring-opening reaction of cyclodextrins (CDs) catalyzed by amylolytic enzymes. In this study, we analyze the catalytic conversion of α-, β-, and γ-CDs by a GH57 family amylopullulanase from Aquifex aeolicus (AaApu) using thin-layer chromatography (TLC). Our findings demonstrate that AaApu has a substrate specificity for γ-CD, while all three CDs exert competitive inhibition on pullulan hydrolysis. To elucidate the molecular mechanism of CDs as inhibitor and substrate of amylopullulanase, we determined high-resolution crystal structures of AaApu (wild-type and D352N) in complex with α-, β-, and γ-CD through co-crystallization. These findings establish a structure–function framework for understanding the bifunctional nature of CDs as both substrates and inhibitors in GH57 amylopullulanases.