Redirecting RiPP Biosynthetic Enzymes to Proteins and Backbone-Modified Substrates

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.

In Vivo Production of Diverse β‐Amino Acid‐Containing Proteins

“…This makes proteins with a wide range of non‐canonical terminal and internal functionalisations accessible. A recent publication [14] also reports protein backbone cyclization by RiPP maturases to form five‐membered heterocycles, suggesting that further peptide maturases might be adaptable to proteins. In addition to their extended carbon backbone, ketoamide units may introduce protease inhibitor pharmacophores and electronically distinct reaction sites for artificial enzymes and orthogonal modifications, thus offering diverse avenues for in vivo and in vitro applications.…”

“…[8,9] While backbone modifications are known from synthetic molecules as well as non-ribosomal and ribosomally synthesized peptides, [10][11][12][13] few natural examples exist for proteins, all of them preserving the α-amino acid topology. [1,14] We recently reported tyramine excision, an unusual modification for ribosomally synthesized and posttranslationally modified peptides (RiPPs) that introduces α-keto-βamino acid residues into the cyanobacterial ca. 100 aa ribosomal precursors PlpA3 and PlpA2.…”

In Vivo Production of Diverse β‐Amino Acid‐Containing Proteins

“…α‐Keto‐β‐amino acid moieties occur in various natural and synthetic bioactive compounds [4] that typically target proteases [5–7] including M pro in SARS‐CoV virus [8, 9] . While backbone modifications are known from synthetic molecules as well as non‐ribosomal and ribosomally synthesized peptides, [10–13] few natural examples exist for proteins, all of them preserving the α‐amino acid topology [1, 14] …”

“…[8,9] While backbone modifications are known from synthetic molecules as well as non-ribosomal and ribosomally synthesized peptides, [10][11][12][13] few natural examples exist for proteins, all of them preserving the α-amino acid topology. [1,14] We recently reported tyramine excision, an unusual modification for ribosomally synthesized and posttranslationally modified peptides (RiPPs) that introduces α-keto-βamino acid residues into the cyanobacterial ca. 100 aa ribosomal precursors PlpA3 and PlpA2.…”

Control of Nucleophile Chemoselectivity in Cyanobactin YcaO Heterocyclases PatD and TruD

“…Their later proteolytic cleavage enables scarless synthesis of diverse, bioactive peptides using highly conserved RSs and biosynthetic enzymes. ,,, Indeed, because of these properties, cyanobactin heterocyclases including PatD, TruD, and LynD are unusually promiscuous and have been widely used in the synthesis of thousands of diverse compounds, − including those with nonproteinogenic analogs of Ser, Thr, and Cys residues (Figure B). Recently, these enzymes have even been used in the context of whole proteins to install heterocycles …”