Computational design of a Diels–Alderase from a thermophilic esterase: the importance of dynamics

A novel computational Diels–Alderase design, based on a relatively rare form of carboxylesterase from Geobacillus stearothermophilus , is presented and theoretically evaluated. The structure was found by mining the PDB for a suitable oxyanion hole-containing structure, followed by a combinatorial approach to find suitable substrates and rational mutations. Four lead designs were selected and thoroughly modeled to obtain realistic estimates of substrate binding and prearrangement. Molecular dynamics simulations and DFT calculations were used to optimize and estimate binding affinity and activation energies. A large quantum chemical model was used to capture the salient interactions in the crucial transition state (TS). Our quantitative estimation of kinetic parameters was validated against four experimentally characterized Diels–Alderases with good results. The final designs in this work are predicted to have rate enhancements of ≈10 3 –10 6 and high predicted proficiencies. This work emphasizes the importance of considering protein dynamics in the design approach, and provides a quantitative estimate of the how the TS stabilization observed in most de novo and redesigned enzymes is decreased compared to a minimal, ‘ideal’ model. The presented design is highly interesting for further optimization and applications since it is based on a thermophilic enzyme ( T opt = 70 °C).