Fast Knoevenagel Condensations Catalyzed by an Artificial Schiff-Base-Forming Enzyme

摘要

The simple catalytic motifs utilized by enzymes created by computational design and directed evolution constitute a potentially valuable source of chemical promiscuity. Here we show that the artificial retro-aldolase RA95.5-8 is able to use a reactive lysine in a hydrophobic pocket to accelerate promiscuous Knoevenagel condensations of electron-rich aldehydes and activated methylene donors. Optimization of this activity by directed evolution afforded an efficient enzyme variant with a catalytic proficiency of 5 × 10~(11) M~(-1) and a ＞10~8-fold catalytic advantage over simple primary and secondary amines. Divergent evolution of de novo enzymes in this way could be a promising strategy for creating tailored biocatalysts for many synthetically useful reactions.