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.