Boron Designer\nEnzyme with a Hybrid Catalytic Dyad

Genetically encoded noncanonical amino acids can introduce\nnew-to-nature\nactivation modes into enzymes. While these amino acids can act as\ncatalysts on their own due to their inherent chemical properties,\ninteractions with adjacent residues in an enzyme, such as those present\nin natural catalytic dyads or triads, unlock a higher potential for\ndesigner enzymes. We incorporated a boron-containing amino acid into\nthe protein scaffold RamR to create an active enzyme for the kinetic\nresolution of α-hydroxythioesters. We found that a closely positioned\nlysine residue is crucial for the catalytic activity of the designer\nenzyme by forming a hybrid catalytic dyad with the boronic acid residue.\nThe enzyme is capable of resolving differently substituted α-hydroxythioesters\nwith good selectivities. High-resolution mass spectrometry, <sup>11</sup>B NMR spectroscopy, and crystal structure analysis of the designer\nenzyme gave insight into the three steps of the mechanism (substrate\nbinding, hydroxide transfer, product release). Mutations of a residue\naround the catalytic dyad led to a variant of the enzyme with 2-fold\nimprovement of catalytic activity and selectivity.