Figure 5. The free energy profile and optimized structures of important transition states (TSs) of the epimerization reaction catalyzed by thioesterase NocTE. The direct deprotonation path and re-protonation path Path1 were colored in pink and green respectively, while the optimal reaction pathway was shown in blue. The important residues (D1806, H1808 and H1901) and LSub were colored in gray and pink respectively.
In the deprotonation step, LSub provided the proton Hα for H1901 straightway with the energy barrier of 16.8 kcal/mol, while the energy barrier for the indirect path with the assistance of the water molecule WatA was 12.4 kcal/mol, 4.4 kcal/mol lower than the former. Combining abundant water molecules observed in MD simulations and energy barrier superiority, the indirect path was considered as the optimal reaction path for the deprotonation. After that, the proton accepted by H1901 was transferred to the O1 atom of LSub to form an enol intermediate (IM2), facilitating the proton movement towards the si face. This process required 10.4 kcal/mol relative to the IM1.
In the re-protonation step, the β-lactam ring played a crucial role in the proton transfer. WatB was anchored around the reaction site by the backbone of H1808 and β-lactam ring in advance. In IM2, WatB formed hydrogen bonds with the β-lactam ring and the enol group of LSub (Figure S8), implying the existence of Path1 and Path2. For Path1, the proton deposited on the LSub was transferred to WatB, synchronizing with the proton transfer from WatB to the Cα atom of LSub, inverting the stereochemistry of the C-terminal Hpg. The energy barrier for this path was 24.2 kcal/mol relative to IM2, suggesting that Path1 was unfavorable for re-protonation due to the prohibitively high barrier. For Path2, the proton transfer from the O1 atom to the O2 atom of the β-lactam ring took place, followed by the proton transfer from the β-lactam ring to the atom Cα, which were both mediated by WatB. The former as the rate-determining step needed 14.0 kcal/mol relative to IM2, and the energy barrier for the latter was 0.3 kcal/mol relative to IM3. Although Path2 seemed more complicated than Path1, the remarkable energy superiority made Path2 more suitable for the re-protonation.
From the view of structural characteristics, the O2 atom of the β-lactam ring got closer to the proton H2 of WatB than the Cα atom in IM2. The C-terminal Hpg and WatB formed a tortuous six-membered structure in the transition state TS5 for Path1, resulting from the steric hindrance of the hydroxyphenyl group and the vertical plane conformation formed by the carbonyl group of the C-terminal Hpg. The O1 atom and the Cα atom stayed too close, leading to no enough space for two proton transfers. Though the similar structure appeared in the transition state TS4 for Path2, the β-lactam ring facilitated the formation of more reasonable horizontal plane for electron delocalization, lowering the energy barrier. Besides, the O2 atom of the β-lactam ring that served as the proton donor got away from the Cα atom, making room for proton transfers. In addition, the imidazole ring of H1808 was found to form hydrogen bonds and π-π stacking interactions with the C-terminal Hpg of LSub to maintain the substrate conformation during the overall reaction, partially elucidating the adverse effect of H1808 mutation on the diastereomer purity of products.
In brief, the indirect protonation step and the substrate-assisted re-protonation step (Path2) composed the optimal reaction pathway for the epimerization catalyzed by NocTE with the total energy barrier of 20.3 kcal/mol, in harmony with experiment results5. The water molecules involved in epimerization were a hint that D-configured product was hard inverted into L-configured products once again, resulting in the stereochemical selectivity of NocTE.