Expanding the Chemical Space of Synthetic Cyclic Peptides Using a Promiscuous Macrocyclase from Prenylagaramide Biosynthesis

Abstract

Cyclic peptides are excellent drug candidates, placing macrocyclization reactions at the apex of drug development. PatG and related dual-action proteases from cyanobactin biosynthesis are responsible for cleaving off the C-terminal recognition sequence and macrocyclizing the substrate to provide cyclic peptides. This reaction has found use in the enzymatic synthesis of diverse macrocycles. However, these enzymes function best on substrates that terminate with the non-proteinogenic thiazole/thiazoline residue, complicating synthetic strategies. Here, we biochemically characterize a new class of PatG-like macrocyclases that natively use proline, obviating the necessity of additional chemical or biochemical steps. We experimentally define the biochemical steps involved in synthesizing the widespread prenylagaramide-like natural products, including macrocyclization and prenylation. Using saturation mutagenesis, we show that macrocyclase PagG and prenyltransferase PagF are highly promiscuous, producing a library of more than 100 cyclic peptides and their prenylated derivatives in vitro. By comparing our results to known cyanobactin macrocyclase enzymes, we catalog a series of enzymes that collectively should synthesize most small macrocycles. Collectively, these data reveal that, by selecting the right cyanobactin macrocyclase, a large array of enzymatically synthesized macrocycles are accessible.

A roadmap for metagenomic enzyme discovery

“…Biosynthetic pathways for the cyanobactins, a class of cytotoxic RiPPs including the patellamides and trunkamides, were originally discovered from metagenomic eDNA. [254][255][256] The promiscuity of RiPP maturases encoded in cyanobactin pathways, e.g., heterocyclases for azoline installation 257 or macrocyclases for cyclic peptide formation, 258 have been exploited to generate libraries of synthetic peptides.…”

Genome‐Mining‐Based Discovery of the Cyclic Peptide Tolypamide and TolF, a Ser/Thr Forward O‐Prenyltransferase

“…A significant subset of cyanobactins are tailored by the F‐type prenyltransferases, which append an isoprene unit to Ser, Thr, Tyr and Trp residues or at the N‐ or C‐terminus of linear peptides [6b,c, 8] . These prenyltransferases have the same secondary structural motifs as ABBA type enzymes with a barrel of 10 anti‐parallel β‐sheets surrounded by a ring of 10 solvent exposed α‐helices [9] .…”

Biocatalytic synthesis of peptidic natural products and related analogues

“…Indeed, PatG-type macrocyclases have synthesized libraries encoding millions of cyanobactin analogues with a tremendous sequence diversity in E. coli ( Ruffner et al., 2015 ). Furthermore, the Schmidt group recently synthesized over 100 cyclic proline-containing cyanobactin peptide analogues and their prenylated derivatives in vitro using the excised macrocyclase domain of PagG and the prenyltransferase PagF ( Sarkar et al., 2020 ). These examples showcase the biocatalytic applications of PatG-type macrocyclases for the synthesis of cyclic peptides.…”

“…Chemical synthesis of a number of RiPP substrates with both natural and unnatural amino acids can be achieved with the SPPS method, and promiscuous biosynthetic enzymes then take these substrates to synthesize peptide libraries. One notable example of this chemoenzymatic strategy is the generation of cyanobactin libraries using several cyanobactin biosynthetic enzymes, e.g., cyclases, heterocyclases, and prenyltransferases ( Oueis et al., 2015 ; Sarkar et al., 2020 ) ( Figure 7 A). For example, the Naismith group chemically synthesized multiple cyanobactin substrates ended with a Cys or Pro residue and followed with a short C -terminal RS, some of which also contain an unnatural amino acid azidoalanine or dehydroalanine.…”