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

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Difructose dianhydride I synthase/hydrolase (αFFase1) is a member of the family 172 of glycoside hydrolases which catalyzes the reversible transformation of inulobiose into difructose dianhydride I (DFA I). This transformation was hypothesized to go through a double-displacement mechanism formed by a glycosylation step and a cyclization step. During the glycosylation step, E270 protonates the O2 of the α-D-fructofuranosyl group in the subsite –1 (–1 sugar), and E291 attacks the anomeric carbon forming a glycosyl-enzyme intermediate (GEI) and releasing a water molecule. During the cyclization, activated by a complex torsional mechanism, the O1 of the β -D-fructofuranosyl group in the subsite +1 attacks the anomeric carbon, cleaving the glycosyl-E291 bond and transferring a proton to the E270. Using hybrid QM/MM metadynamics methods, we demonstrate the energetic accessibility of the glycosylation + cyclization mechanism for the αFFase1-inulobiose complex. Our simulations show a slightly exothermic global reaction (∆G 0 = –1.3 kcal·mol−1) in good agreement with the experimental inulobiose/DFA I resulting ratios (1:8.9). The ∆G ‡ and ∆G 0 for the glyco- sylation are 12.3 and –2.8 kcal·mol−1. The rate-limiting step is the cyclization with a ∆G ‡ = 15.3 kcal·mol−1. Our study shows that the –1 sugar follows the E5 → E5/4T5 → E3 and E3 → 4E → 4E conformational pathways. Further analysis of the cyclization simulation shows the role of E85, K147, and N226 stabilizing the rotation of the +1 sugar and facilitating the attack of the O1’ to the anomeric carbon.