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.