Rational Design of Dehydrogenase/Reductases Based on Comparative Structural Analysis of Prereaction-State and Free-State Simulations for Efficient Asymmetric Reduction of Bulky Aryl Ketones

Inspired by the conformational change of the enzyme-substrate complex in molecular dynamics (MD) simulation with distance restriction, we propose a strategy for identifying the engineering targets based on the comparative analysis of enzyme-/substrate- binding modes in MD simulations with and without distance restriction (prereaction-state simulation and free-state simulation). Taking the short-chain dehydrogenase/reductase (SDR) mutant EbSDR8-G94A/S153L (Mu0) with poor activity toward bulky aryl ketones as an example, H145 and Y188 were identified as the engineering targets due to the distinct conformation difference in the two simulation modes. To break the beam structure formed by these residues at the entry of cavity C2 in free-state simulation, the substrate-binding pocket was reconstructed, and meanwhile the relative size of cavities C1 and C2 was modulated to improve the enantioselectivity. In this way, mutants for efficient asymmetric reduction of o-halogenated acetophenones, propiophenones, aromatic ketoesters, and diaryl ketones were designed, delivering chiral alcohols with >99% conversion and >98% ee. The effectiveness of this design strategy was also validated by the successful redesign of PpYSDR, generating a variant for efficient reduction of (4-chlorophenyl) 2-pyridyl ketone into the S-product with >99% conversion and 96% ee. MD simulations suggested a suitable binding pocket with proper energy contribution as the ubiquitous mechanism for the improvement of activity and enantioselectivity toward substrates with varied structures. The success in this study provides hints for the rational design of alcohol dehydrogenases/reductases with both a broad substrate spectrum and high enantioselectivity.