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

The difructose dianhydride I synthase/hydrolase $\alpha \mathrm{FFase}1$ 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 $\alpha \mathrm{FFase}1$ 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 ${}_5$ $\to$ E ${}_5$ / ${}^4$ T ${}_5$ $\to$ E ${}_3$ $\to$ ${}^4$ 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 $\alpha \mathrm{FFase}1$ variant. Enzyme activity data confirm an important role for N226 during the cyclization step but show that it is dispensable for the hydrolysis step.