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.