QM/MM Theoretical Studies of a de Novo Retro-Aldolase Design

The design of innovative enzymes is a standing goal to obtain original specific catalysts to work under mild conditions of temperature and pressure. Attempts to get artificial enzymes become particularly difficult when the target is a reaction proceeding in a multistep mechanism such as the Retro-Aldol reaction. The goal of this work is to study the reaction mechanism of the most efficient de novo retro-aldolase design, the RA95.5-8F, and to understand the origin of its catalytic power. Our theoretical studies have been based on the analysis of free-energy surfaces employing hybrid QM/MM molecular dynamics simulations, with the QM subset of atoms described by semiempirical (AM1) and DFT (M06-2X) methods. The complete free-energy landscape of the reaction, generated in terms of potentials of mean force for each step, suggest that the rate-limiting step corresponds to the decomposition of an enamine intermediate into a Schiff base. This result agrees with the experimental data. Computed inverse secondary deuterium kinetic isotope effects (2° KIEs) are also in agreement with steady-state kinetic experiments. A detailed description of the reaction mechanism at the molecular level can pave the way to propose mutations that could enhance the activity of this complex multistep catalyzed process by proposing mutations that would stabilize the transition-state structures appearing along the reaction