Figure 1. (A) The epimerization-hydrolysis catalytic activities
of bifunctional thioesterase NocTE; (B) The structures of the precursor
peptide, nocardicin G and nocardicin A.
The hydrolytic activity of NocTE is mediated by catalytic triad
S1779-H1901-D1806. Once the substrate is loaded on the serine (S1779) of
NocTE, the histidine (H1901) captures a proton from a water molecule
near the active site and promotes the remaining
nucleophile to attack the substrate,
forming a tetrahedral hydrolytic intermediate. Then the covalent bond
between the substrate and S1779 breaks and the product gradually escapes
from the active pocket. At the same time, H1901 returns the proton
towards S1779 to restore the initial enzymatic environment, ready for
the next catalysis cycle. However, the epimerization catalyzed by
thioesterases has not been studied so far. As we know, two catalytic
residues are usually the requisite for epimerization reaction, one of
which acts as the base to abstract the proton (deprotonation) and then
the other (maybe a water molecule) complements a proton
(re-protonation).4, 11-13 For the mysterious
epimerization activity of NocTE, Andrew M. Gulick et
al. 6 has proposed that the histidine H1901 of the
catalytic triad deprotonates the substrate according to site-directed
mutagenesis results and crystal architecture characteristics, while the
detailed catalytic mechanism is still ambiguous. Although the catalytic
activities of S1779 mutants and H1901 mutants are completely lost, it is
undistinguishable whether these mutations mainly effect the hydrolysis
or epimerization reaction. In addition, H1808 mutant can catalyze the
generation of a few L-diastereomers (epi -nocardicin G,
D-Hpg-L-β-lactam-L-Hpg), influencing the stereochemical selectivity of
NocTE. More detailed information about NocTE needs further
investigations, such as how the epimerization reaction proceeds, how the
epimerization and hydrolysis combine, and why epi -nocardicin G is
unable to hydrolyze efficiently before the stereochemical inversion.
In this study, we investigated the catalytic mechanism and
stereochemical selectivity of the thioesterase NocTE through a series of
computational methods. The pre-reaction structures for epimerization
were sampled by analyzing the distributions of deprotonation
conformations. Combining structural characteristics abstracted from MD
simulations, the molecular mechanism of the substrate-assisted
epimerization was proposed and confirmed by QM/MM calculations. Besides,
the energy barrier difference between hydrolysis reactions forepi -nocardicin G and nocardicin G further explained the
stereochemical selectivity for NocTE catalysis. Our studies uncover the
catalytic mechanism of bifunctional thioesterase NocTE and generate a
spark of appreciation for the research and development of novel peptide
drugs.