Structural analysis of (2 → 1)-β-d-fructofuranosides linked to a terminal difructose dianhydride III produced by Bacteroides endo-type inulin fructotransferase.

The glycoside hydrolase (GH) family 91 inulin fructotransferase (IFTase) complexes from Bacteroides ovatus and B. caccae act as endo-type IFTases targeting inulin. However, their degradation mechanism remains unclear. Herein, the exact structure of the accumulated inulin-degradation product in a culture supernatant is revealed as linear oligo-(2 → 1)-β-d-fructofuranosides linked to difructose dianhydride III (DFA III) at the reducing end. Additionally, we developed a method to quantify endo-IFTase activity by measuring DFA III released from inulin after sequential treatment with endo-IFTase and GH32 β-d-fructofuranosidase. Using this approach, we investigated the effect of varying concentrations of endo-IFTase subunits 1 and 2 and found that an equimolar mixture of the two subunits exhibited the highest enzymatic activity, indicating that the active complex forms in a 1:1 ratio. The endo-IFTase accepts fructooligosaccharide DP7 (GF) as the shortest substrate, suggesting that the complex recognizes the region between subsites +3 and - 3. This study provides insights into the understanding of inulin degradation by Bacteroides species and elucidates the molecular mechanisms underlying prebiotic effects of inulin.