Integrating noncanonical amino acids and synthetic cofactors for the development of artificial metalloenzymes

Natural metalloenzymes harness the sophisticated synergy between canonical amino acids and metal cofactors to catalyze life-sustaining reactions. However, the limited chemical diversity of the twenty standard amino acids often restricts non-natural transformations. To overcome these constraints, artificial metalloenzymes (ArMs) integrate synthetic cofactors into protein scaffolds to enable unnatural catalytic functions. This review highlights how noncanonical amino acids (ncAAs) bridge the gap between natural enzyme architecture and synthetic chemistry. We examine genetic code expansion and codon optimization technologies as robust strategies for the site-specific incorporation of ncAAs. By introducing unique chemical moieties—such as chelating groups, photo-responsive elements, and bio-orthogonal handles—ncAAs enable precise mechanistic probing of active sites and the rational design of biomimetic centers. Furthermore, we discuss the strategic use of ncAAs in anchoring metal ions and large-sized synthetic cofactors via bio-orthogonal reactions, allowing for the creation of hybrid catalysts that maintain structural integrity while exhibiting novel reactivity. Ultimately, the combinatorial design of ncAAs and synthetic cofactors provides a transformative toolkit for protein engineering. Integrating these structural elements transcends natural limitations, laying a foundation for ArMs with enhanced catalytic efficiency, expanded substrate scope and programmable activity for industrial and therapeutic applications.