GH172 difructose dianhydride I synthase: A two-stepped journey via glycosylation and cyclization using QM/MM metadynamics

The difructose dianhydride I synthase/hydrolase αFFase1 catalyzes the reversible transformation of inulobiose into difructose dianhydride I, a compound present in differ ent caramel compositions. The proposed reaction mechanism of such a transformation consists of a glycosylation step followed by a cyclization to yield the dianhydride from the disaccharide. In response to the absence of mechanistical details for the reaction, we made use of extensive hybrid QM/MM metadynamics studies to shed light on the molecular basis of it. Our simulations show that the global reaction for the transfor mation of inulobiose by αFFase1 is slightly exothermic (∆G0 =–1.3 kcal mol−1) in good agreement with the experimental data. Furthermore, we observe that the glyco sylation step is the rate-limiting step of the reaction with an energy barrier of 12.3 kcal mol−1 compared to 8.7 kcal mol-−1 for the cyclization step. Interestingly, our work shows that the–1 sugar of inulobiose changes from an initial E5 conformation into a 4E puckering via the conformational pathway E5 → E5/4T5 → E3 → 4E. Our calculations highlight that in the cyclization step, the three residues E85, K147, and N226 are es sential for the rotation of the +1 sugar in the substrate to facilitate the intramolecular attack of oxygen O1’ to the anomeric carbon. Prompted by these results, we expressed and assayed the N226A αFFase1 variant. Kinetic data confirm an important role for N226 during the cyclization step but are irrelevant for the hydrolysis step.