title: Fast Knoevenagel Condensations Catalyzed by an Artificial Schiff-Base-Forming Enzyme.
abstract: 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.
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich , 8093 Zurich, Switzerland.
authors.forename: Xavier
authors.initials: X
authors.lastname: Garrabou
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich , 8093 Zurich, Switzerland.
authors.forename: Basile I M
authors.initials: BI
authors.lastname: Wicky
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich , 8093 Zurich, Switzerland.
authors.forename: Donald
authors.initials: D
authors.lastname: Hilvert
mesh_terms: D000447:Aldehydes / Q000737:chemistry*; D055162:Biocatalysis; D019295:Computational Biology; D019020:Directed Molecular Evolution; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry*; D008958:Models, Molecular; D015394:Molecular Structure; D012545:Schiff Bases / Q000737:chemistry
chemical_list: D000447:Aldehydes; D012545:Schiff Bases; D005634:Fructose-Bisphosphate Aldolase; D008239:Lysine