Simultaneously, as the hydride migrates, C6 gradually approaches the double bond C2=C3, facilitating the formation of a C1-C2-C6 three-membered ring motif, ultimately yielding C + state with a positive charge localized at C3. This C + is stabilized by a cationπ interaction with F93, exhibiting an interaction energy of 4.2 kcal/mol. This concerted hydride transfer and cyclization process require overcoming a slight energy barrier of 2.4 kcal/mol, accompanied by a substantial heat release of 14.6 kcal/mol (Supplementary Figure ), presumably stemming from the enhanced stability achieved the reformation of the three-membered ring motif. It should be noted that the C3 bears the most positive charge while C6 also exhibits a partial charge accumulation, with respective atomic charges of 0.27 and 0.17. As a result, the hydrogen atom (Hc) located at C1 could transfer to C6, thereby destructing the three-membered ring and constructing the 6,6-bicyclic structure, resulting in a D + state (Figs. and). The above transition step needs overcoming a barrier of 11.9 kcal/mol, accompanied by a heat release of 9.9 kcal/mol (Supplementary Figure ). In D + , the vacant p-orbital of C1 conjugates with C2=C3, distributing the positive charge between C1 and C3. While considering an alternative pathway for the formation of D + , a direct transition from B + to D + via 1,3 hydride migration (from C1 to C6) seems plausible. However, the hydrogen atoms on C1 and C6 are positioned on opposing sides of the rigid ring in C + , thereby hindering a direct transfer to C6 due to unfavorable spatial orientation (Supplementary Figure ). In D + , the hydrogen (Hd) positioned on C16 is in close proximity to the oxygen atom (O1) of OPP, with a distance of 2.0 Å (Supplementary Figure ), accordingly, the basic O1 abstracts Hd, generating the ultimate product 3 (Figs. and). This transformation involves a tiny barrier of 1.3 kcal/mol and exhibits a notable exothermicity of 20.5 kcal/mol (Supplementary Figure ). We next implemented mutational studies (Fig. ) based on the structural information from crystal experiments and mechanism insights from QM/MM calculations. We first mutated residues interacting with the pyrophosphate group, including C153, K196, R212, D215, S270, and K266. Notably, R212, D215, and S270 were identified as critical for activity. Notably, mutating K196 to A, R, or T partially restored activity, indicating the influence of the amino acid's acidity on its interaction with the pyrophosphate group, whereas mutations to D or E were ineffective.