A Versatile Enzymatic Pathway for Modification of Peptide C‑Termini

Advances in bioinformatics
have enabled the discovery
of unique
enzymatic reactions, particularly for ribosomally synthesized and
post-translationally modified peptides (RiPPs). The recently discovered
daptides, peptides with their C-terminus replaced by an amine, represent
one such case, but the diversity, requirements, and engineering potential
of daptide biosynthesis remain to be established. Using the daptide
biosynthetic gene clusters from Thermobifida fusca and Streptomyces azureus, we reconstituted daptide
biosynthesis in vitro, revealing the enzymatic requirements
for successive oxidative decarboxylation, transamination, and N,N-dimethylation. In vitro and in vivo studies showed a tailoring family of
YcaO enzymes convert a secondary amine intermediate to a C-terminal
imidazoline. We further demonstrated enzymatic activity toward shortened,
leader peptide-free, and non-native core peptides, highlighting a
broad substrate tolerance. Using these insights, we directed the daptide
pathway to install new C-termini, including a bioconjugation-compatible
aminoacetone, on various peptide and protein substrates.