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.