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 de...