Robust design and optimization of retroaldol enzymes

TLDR

The authors computationally designed retroaldol enzymes by incorporating a lysine in a nonpolar environment and water‑mediated stabilization of carbinolamine and β‑hydroxyl groups. The design pipeline produced 33 active enzymes on 13 scaffolds, with activity boosted by directed mutagenesis and evolution; mutational analysis highlighted improvement hotspots, and distinct catalysts generated varied borohydride‑reduced intermediates, revealing a diverse property landscape.

Abstract

Abstract Enzyme catalysts of a retroaldol reaction have been generated by computational design using a motif that combines a lysine in a nonpolar environment with water‐mediated stabilization of the carbinolamine hydroxyl and β‐hydroxyl groups. Here, we show that the design process is robust and repeatable, with 33 new active designs constructed on 13 different protein scaffold backbones. The initial activities are not high but are increased through site‐directed mutagenesis and laboratory evolution. Mutational data highlight areas for improvement in design. Different designed catalysts give different borohydride‐reduced reaction intermediates, suggesting a distribution of properties of the designed enzymes that may be further explored and exploited.