Mechanistic study of the biosynthesis of R-phenylcarbinol by acetohydroxyacid synthase enzyme using hybrid quantum mechanics/molecular mechanics simulations

The biosynthesis of R-phenylacetylcarbinol (R-PAC) by the acetohydroxy acid synthase, (AHAS) is addressed by molecular dynamics simulations (MD), hybrid quantum mechanics/molecular mechanics (QM/MM), and QM/MM free energy calculations. The results show the reaction starts with the nucleophilic attack of the C2α atom of the HEThDP intermediate on the Cβ atom of the carbonyl group of benzaldehyde substrate via the formation of a transition state (TS1) with the HEThDP intermediate under 4′-aminopyrimidium (APH+) form. The calculated activation free energy for this step is 17.4kcal mol−1 at 27 °C. From this point, the reaction continues with the abstraction of Hβ atom of the HEThDP intermediate by the Oβ atom of benzaldehyde to form the intermediate I. The reaction is completed with the cleavage of the bond C2α-C2 to form the product R-PAC and to regenerate the ylide intermediate under the APH+ form, allowing in this way to reinitiate to the catalytic cycle once more. The calculated activation barrier for this last step is 15.9kcal mol−1 at 27 °C. [Display omitted] •The biosynthesis of R-PAC by AHAS is addressed for first time by quantum mechanics/molecular mechanics simulations.•The mechanism consist of two stages, namely the attack of HEThDP on benzaldehyde, and the cleavage of the C2α-C2 bond.•The calculated free activation energies for the first and second stages are 16 kcal/mol and 14 kcal/mol, respectively.•The formation of R-PAC competes directly with the formation of the physiological product acetolactate.•The HEThDP intermediate is in its 4'-aminopyrimidium (APH+) form during the reaction pathway.