Redirecting RiPP Biosynthetic Enzymes to Proteins and Backbone-Modified Substrates

Joshua A Walker; Noah Hamlish; Avery Tytla; Daniel D Brauer; Matthew B Francis; Alanna Schepartz

doi:10.1021/acscentsci.1c01577

Redirecting RiPP Biosynthetic Enzymes to Proteins and Backbone-Modified Substrates

ACS Cent Sci. 2022 Apr 27;8(4):473-482. doi: 10.1021/acscentsci.1c01577. Epub 2022 Mar 21.

Authors

Joshua A Walker^{1

2}, Noah Hamlish^{3

2}, Avery Tytla¹, Daniel D Brauer^{1

2}, Matthew B Francis^{1

4

2}, Alanna Schepartz^{1

3

4

2}

Affiliations

¹ Department of Chemistry, University of California, Berkeley, California 94720, United States.
² Center for Genetically Encoded Materials, University of California, Berkeley, California 94720, United States.
³ Department of Molecular and Cellular Biology, University of California, Berkeley, California 94720, United States.
⁴ California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, California 94720, United States.

Abstract

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.