To exclude the carboxylation step (see Figure ), the QM region only included the reactive portions of the substrate crotonyl-CoA and NADPH (see the CCR scheme of Figure ), being reduced compared to our previous study. The link-atom approach was used to saturate missing valences at the QM/MM interface, resulting in a total of 48 QM atoms (two hydrogens as link-atoms) with a net charge of zero. QM/MM simulations utilized both original and optimized parametrizations of the GFN2-xTB semiempirical method. The MM region was treated with the CHARMM36 force field and parameters derived for NADPH co-factor and crotonyl-CoA substrate. Periodic Boundary Conditions (PBC) were employed for both QM and MM regions, combined with 8Å cutoffs in direct space.