Expanding enzyme functionality through site-specific incorporation of noncanonical amino acids.

Enzymes are nature's catalysts, capable of performing complex chemical transformations with remarkable precision and efficiency, yet their potential is confined by the chemistry of 20 canonical amino acids. The advent of genetic code expansion has unlocked a powerful route to transcend this limitation by enabling the site-specific incorporation of noncanonical amino acids (ncAAs) with novel chemical functionalities. Once primarily used as mechanistic probes, ncAAs are now emerging as transformative design elements in enzyme engineering, reshaping the electronic and structural landscape of active sites and introducing novel catalytic capabilities with finely tuned selectivity. This review highlights rapidly advancing frontiers in metalloenzymes, organocatalytic enzymes, and photoenzymes, where ncAAs offer innovative strategies to modulate catalysis, broaden reaction scope, and enhance enzymatic function.