Artificial Enzyme Design with Novel Functions via Site‐Specific Chemical Modification

The design of artificial enzymes represents a transformative advancement in biocatalysis, enabling the creation of bespoke biocatalysts for nonnatural reactions. A key innovation in this field is the introduction of unnatural catalytic residues through site-specific chemical modification, which significantly expands the chemical repertoire of natural enzymes. This approach combines precision engineering with cutting-edge methodologies, including chemical ligation, noncanonical amino acid incorporation and directed evolution. These strategies facilitate the development of enzymes with novel catalytic activities, modify substrate specificities, and enhance stability under nonphysiological conditions. This concept examines the methodologies, challenges, and future directions in the design of enzymes with unnatural catalytic residues via site-specific chemical modification, with a focus on their functional impact and transformative potential in synthetic chemistry and biocatalysis.