Structural and Functional Basis of Difructose Anhydride Ill Hydrolase, Which Sequentially Converts Inulin Using the Same Catalytic Residue

Fructan inulin is composed of polymeric fructoses linked by O-glycosidic bonds, and a variety of enzymes are involved in its decomposition to provide energy for organisms. Specifically, inulin fructotransferase (IFTase) depolymerizes inulin to difructose anhydride III (DFA-III). DFA-III was reported to be further degraded by DFA-III hydrolyase (DFA-Mase). This work reveals that the structure of DFA-IIIase is a trimer, with each monomer displaying a right-handed beta-helix fold, which resembles IFTase except for an extra lid covering the active center. With this lid, DFA-IIIase is capable of converting inulin to DFA-III (IFTase activity) in addition to hydrolyzing DFA-III using the same site and reaction conditions. This unusual and unexpected sequential catalysis is ascribed to the extremely conserved residues in the active center of IFTase and DFA-Illase and the protonated states of the catalytic residue that are regulated by the opening and closing of the lid. This work paves the way for further investigation of the metabolism of inulin in nature and provides an example of sequential enzymatic catalysis.