3.4 The mechanism of hydrolysis reaction
After the epimerization, the substrate with the D-Hpg in the C-terminal (DSub) should be released via hydrolysis reaction. The system of nocardicin G covalently bound to NocTE (NocTE-DSub) was modeled to show the energy profile of hydrolysis. The starting conformation for QM/MM calculation was the representative structure from the pre-reaction states in six replicas of 50 ns MD simulation trajectories (Figure S9). The free energy profile and key minimum structures for hydrolysis were shown in Figure 6A, Figure S10 and Table S6. Some important distances involved in the reaction were listed in Table S7.
As we can see, H1808 still helped DSub to maintain the proper hydrolytic conformation through hydrogen bonding and π-π stacking interactions. Firstly, H1901 abstracted a proton from the water molecule WatC to promote the nucleophilic attack on the C1 atom, generating a tetrahedral intermediate IM. The energy barrier for this step was 13.5 kcal/mol relative to the reactant R. Subsequently, the Oγ-C1 bond broke and H1901 provided the proton for S1779, resulting in the product generation and enzymatic environment recovery for the next catalysis cycle. The energy barrier for the whole hydrolysis process was 14.3 kcal/mol, basically consistent with the fact that the rate of hydrolysis was faster than that of epimerization8.