In pursuit of a comprehensive understanding of TSs' catalytic mechanism, QM/MM calculations have emerged as an effective tool, beyond isotope labeling studies and crystal enzyme structure studies, offering valuable insights into the enzyme's role in stabilizing reaction intermediates, both in this work and previous studies . Our QM/MM calculation revealed the rationality of 2,6-cyclization and opening occurring between two 1,2-hydride shifts prior to final deprotonation, which was evidenced by the production of 11 by PcTS1 F221V . Moreover, our QM/MM calculations facilitated the resolution of identifying the pyrophosphate as the deprotonation base, which is a prediction challenging in our initial mechanism proposal and difficult to validate through labeling experiments. This successful application of QM/MM calculations underscores the importance of an integrative approach encompassing isotope labeling studies, crystal enzyme structure studies, and QM/MM calculations for understanding the catalytic mechanism of TSs.