Redirecting RiPP Biosynthetic Enzymes to Proteins and Backbone-Modified Substrates

Ribosomally synthesized
and post-translationally modified peptides
(RiPPs) are peptide-derived natural products with potent antibiotic,
antiviral, and anticancer properties. RiPP enzymes known as cyclodehydratases
and dehydrogenases work together to catalyze intramolecular, inter-residue
condensation and dehydrogenation reactions that install oxazoline/oxazole
and thiazoline/thiazole heterocycles within ribosomally produced polypeptide
chains. Here, we show that the previously reported enzymes MicD-F
and ArtGox accept backbone-modified monomersincluding aminobenzoic
acid derivatives and beta-amino acidswithin leader-free polypeptides,
even at positions immediately preceding or following the site of cyclization/dehydrogenation.
The products are sequence-defined chemical polymers with multiple,
diverse non-α-amino acid subunits. We show further that MicD-F
and ArtGox can install heterocyclic backbones within protein loops
and linkers without disrupting the native tertiary fold. Calculations
reveal the extent to which these heterocycles restrict conformational
space; they also eliminate a peptide bondboth features could
improve the stability or add function to linker sequences now commonplace
in emerging biotherapeutics. This work represents a general strategy
to expand the chemical diversity of the proteome beyond and in synergy
with what can now be accomplished by expanding the genetic code.