Harnessing P450 enzyme plasticity for generalizable peptide biaryl macrocyclization

Macrocyclic peptides containing biaryl motifs, predominantly derived from natural products, are valuable scaffolds due to their structural rigidity and potent bioactivity. However, current synthetic methods remain constrained by the absence of broadly applicable strategies across chemocatalytic and biocatalytic platforms. Here, we discover a versatile P450 enzyme, GpeC, capable of facilitating oxidative C-C/O/N cross-coupling for peptide biaryl macrocyclization (PBC) with ‘leader-independent’ activity. Crystal structure analysis of GpeC rationalizes leader-independent property and reveals ‘adaptive recognition’ that enables access to diverse biaryl-linked macrocycles. GpeC exhibits exceptional substrate promiscuity, accommodating 19 of 20 canonical amino acids and diverse noncanonical analogs within a minimal tetrapeptide scaffold. The efficient semi-synthesis of the natural product Rubrin further demonstrated the versatility of GpeC. We further introduced ‘lytic to tetraregion’ (LTT), a one-step, single enzyme synthesis for modular synthesis of biaryl-cyclized tetrapeptide. Overall, GpeC’s robustness and programmability position it as a broadly applicable biocatalyst for the synthesis of biaryl macrocyclic peptides.