Protein-derived cofactors: chemical innovations expanding enzyme catalysis

Protein-derived cofactors, formed through posttranslational modification of a single amino acid or covalent crosslinking of amino acid side chains, represent a rapidly expanding class of catalytic moieties that redefine enzyme functionality. Once considered rare, these cofactors are recognized across all domains of life, with their repertoire growing from 17 to 38 types in two decades in our survey. Their biosynthesis proceeds via diverse pathways, including oxidation, metal-assisted rearrangements, and enzymatic modifications, yielding intricate motifs that underpin distinctive catalytic strategies. These cofactors span paramagnetic and non-radical states, including both mono-radical and crosslinked radical forms, sometimes accompanied by additional modifications. While their discovery has accelerated, mechanistic understanding lags, as conventional mutagenesis disrupts cofactor assembly. Emerging approaches, such as site-specific incorporation of non-canonical amino acids, now enable precise interrogation of cofactor biogenesis and function, offering a viable and increasingly rigorous means to gain mechanistic insights. Beyond redox chemistry and electron transfer, these cofactors confer enzymes with expanded functionalities. Recent studies have unveiled new paradigms, such as long-range remote catalysis and redox-regulated crosslinks as molecular switches. Advances in structural biology, mass spectrometry, and biophysical spectroscopy continue to elucidate their mechanisms. Moreover, synthetic biology and biomimetic chemistry are increasingly leveraging these natural designs to engineer enzyme-inspired catalysts. This review integrates recent advances in cofactor biogenesis, reactivity, metabolic regulation, and synthetic applications, highlighting the expanding chemical landscape and growing diversity of protein-derived cofactors and their far-reaching implications for enzymology, biocatalysis, and biotechnology.