Boron Designer Enzyme with a Hybrid Catalytic Dyad

Abstract

Genetically encoded noncanonical amino acids can introduce new-to-nature activation modes into enzymes. While these amino acids can act as catalysts on their own due to their inherent chemical properties, interactions with adjacent residues in an enzyme, such as those present in natural catalytic dyads or triads, unlock a higher potential for designer enzymes. We incorporated a boron-containing amino acid into the protein scaffold RamR to create an active enzyme for the kinetic resolution of α-hydroxythioesters. We found that a closely positioned lysine residue is crucial for the catalytic activity of the designer enzyme by forming a hybrid catalytic dyad with the boronic acid residue. The enzyme is capable of resolving differently substituted α-hydroxythioesters with good selectivities. High-resolution mass spectrometry, 11B NMR spectroscopy, and crystal structure analysis of the designer enzyme gave insight into the three steps of the mechanism (substrate binding, hydroxide transfer, product release). Mutations of a residue around the catalytic dyad led to a variant of the enzyme with 2-fold improvement of catalytic activity and selectivity.