GH172 Difructose Dianhydride I Synthase: A Two-Stepped Journey via Glycosylation and Cyclization Using QM/MM Metadynamics.

The difructose dianhydride I synthase/hydrolase α FFase 1 catalyzes the reversible transformation of inulobiose into difructose dianhydride I, a component of caramel. The proposed reaction mechanism of such a transformation consists of a glycosylation step followed by a cyclization to yield the dianhydride from the disaccharide. Here, we make 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 transformation of inulobiose by α FFase 1 is slightly exergonic in good agreement with the experimental data. Furthermore, we observe that the glycosylation step is the rate-limiting step. Interestingly, our work shows that the -1 sugar of inulobiose changes from an initial E 5 conformation into a 4 E puckering via the conformational pathway E 5 → E 5 / 4 T 5 → E 3 → 4 E. Our calculations highlight that in the cyclization step, the three residues E85, K147, and N226 are essential 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 α FFase 1 variant. Enzyme activity data confirm an important role for N226 during the cyclization step but show that it is dispensable for the hydrolysis step.