2.3 QM/MM calculations
In order to balance the computational cost and quality, cluster models
were constructed to study the reaction energy profile for thioesterase
NocTE via a two-layered QM/MM ONIOM scheme27, 28implemented in Gaussian 09 program.
The
initial conformations originated from the most dominant cluster and
pre-reaction states of MD simulations. In the cluster model, the
residues within 5.00 Å of the substrate were reserved, containing the
substrate (LSub or DSub), 25 residues and several water molecules near
the catalytic center (Figure S1). The atom number for the cluster model
was 444 in total. The backbone of all residues was frozen to preserve
the original protein architecture. The QM region consisted of the
substrate covalently bound to NocTE, the side chains of D1806 and H1901,
the whole residue H1808 and 3 water molecules, 93 atoms in total.
The geometry optimizations for transition states (TSs) and other minima,
and intrinsic reaction coordinate (IRC) calculations were performed at
the ONIOM (M062X/6-31G*:Amber) level. Each transition state structure
was confirmed with a sole imaginary frequency and reasonable movement
tendency. After that, single point energy calculations for each minimum
were carried out using 6-311+ G** basis set and solvation model based on
density (SMD) solvation correction29 to improve the
accuracy of energy profiles. The interaction between QM and MM layers
was treated with the electrostatic embedding
formalism30.