3.3 The mechanism of epimerization reaction
Distinguishable from other epimerases, the main product pool generated by thioesterase NocTE is D-configured peptides (nocardicin G) and no obvious proton donor exists around the active site for epimerization. According to the structural characteristics abstracted from MD simulations, two possible reaction paths were put forward for deprotonation and re-protonation steps respectively. For the deprotonation step, the proton acceptor H1901 can extract the proton Hα from LSub directly (direct path), or the proton is transferred via the water molecule WatA from LSub to H1901 (indirect path) (Figure 4). Then this proton moves from H1901 to the si face with the assistance of the O1 atom of LSub, prepared for the following step. For the re-protonation step, we come up with two possible reaction paths, Path1 and Path2. In Path1, the proton is transferred from LSub to WatB in thesi face and the Cα atom obtains a proton from WatB simultaneously, generating the epimerization product DSub. In Path2, the β-lactam ring facilitates the proton transfer. The proton transfer from O1 atom to the O2 atom of β-lactam ring is mediated by WatB firstly, and then this proton is transferred via WatB from the β-lactam ring towards the Cα atom again.