4 CONCLUSION
In this work, we presented detailed descriptions of catalytic mechanism for the thioesterase NocTE, a promising insight into substrate-assisted epimerization with stereochemical selectivity for directed drug design. Through restrained MD simulations, we sampled enough pre-reaction conformations for epimerization reaction. From the main structural cluster, two water molecules were inferred to promote the proton transfer. Then a substrate-assisted molecular mechanism for epimerization activity was explored and confirmed by QM/MM calculations. The overall free energy barrier of the epimerization mediated by water molecules and β-lactam-ring was obtained to be 20.3 kcal/mol and the energy barrier of the subsequent hydrolysis was calculated to be 14.3 kcal/mol. In addition, we assessed the hydrolysis difficulty of L-configured substrate without epimerization (LSub) and found the energy profile for the nucleophilic attack of water molecules towards LSub was up to 24.0 kcal/mol. The remarkable barrier disparity for DSub and LSub hydrolysis provided more clues to the stereochemical selectivity of NocTE catalysis, in addition to the comparison of product release in our previous studies10.
Two water molecules became the key to promote epimerization, contributing to the preference reflected in the stereochemical inversion. The pathway choice of water molecule WatA attached importance upon the epimerization activity for NocTE catalysis. During the deprotonation step, the direct hydrolysis of LSub was hindered because the hydrolytic site C1 atom was relatively far from WatA in comparison with the epimerization site Cα atom, preventing WatA from approaching C1 atom to participate in hydrolysis. Therefore, after H1901 abstracted a proton from WatA, the proton Hα from the Cα atom could be easily caught by the deprotonated WatA, resulting in the regeneration of WatA and the deprotonation of LSub. Another water molecule WatB was involved in the proton cyclization for overall reaction, making the D to L stereochemical inversion more difficult. WatB was found to stay around the reaction site Cα atom by forming three stable hydrogen bonds with H1808 and the β-lactam ring of LSub for re-protonation. With the assistance of β-lactam ring, WatB delivered a proton to the Cα atom, completing the L to D stereochemical inversion of the C-terminal Hpg.
The role of β-lactam ring was also displayed in the catalytic process. In deprotonation step, the β-lactam ring of LSub helped to maintain the water molecule WatB around the catalytic center by forming a strong hydrogen bond. Besides, the β-lactam ring directly took part in the proton transfer in the following re-protonation step and its coplanar structure was beneficial for electron delocalization to lower the energy barrier, suggesting the significance of the formation of β-lactam ring in advance distinguishable from that in the biosynthesis of isopenicillin N31. Another key residue that should be noticed was H1808. H1808 formed the hydrogen bond and π-π stacking interactions with the hydroxyphenyl group of the C-terminal Hpg to stable the substrate conformation, revealing the importance of H1808 in the control of diastereomeric purity.
Our findings draw a clear picture of the stereochemical selectivity for bifunctional thioesterase NocTE and provide a reasonable molecular mechanism as an example of substrate-assisted catalysis. These results contribute to the diversification of the thioesterase catalytic activities and encourage the stereochemical control for peptide drugs through rational designs.