Biosynthesis of Cittilins, Unusual Ribosomally Synthesized and Post-translationally Modified Peptides from Myxococcus xanthus

Abstract

Cittilins are secondary metabolites from myxobacteria comprised of three L-tyrosines and one L-isoleucine forming a bicyclic tetrapeptide scaffold with biaryl and aryl-oxygen-aryl ether bonds. Here we reveal that cittilins belong to the ribosomally synthesized and post-translationally modified peptide (RiPP) family of natural products, for which only the crocagins have been reported from myxobacteria. A 27 amino acid precursor peptide harbors a C-terminal four amino acid core peptide, which is enzymatically modified and finally exported to yield cittilins. The small biosynthetic gene cluster responsible for cittilin biosynthesis also encodes a cytochrome P450 enzyme and a methyltransferase, whereas a gene encoding a prolyl endopeptidase for the cleavage of the precursor peptide is located outside of the cittilin biosynthetic gene cluster. We

A biaryl-linked tripeptide from Planomonospora reveals a widespread class of minimal RiPP gene clusters

“…Also for 6, the BGC encodes just one cytochrome P450 enzyme and a precursor peptide, the latter, however, containing a 22amino acid leader peptide. Similarly, the recently reported BGC for cittilin A (7), a crosslinked YIYY tetrapeptide from Myxococcus xanthus, encodes a 27-amino acid precursor peptide, an adjacent cytochrome P450 enzyme, a methyltransferase and a distant endopeptidase for cleavage (Hug et al, 2020). Cytochrome P450 monooxygenases are known to install crosslinks between aromatic residues of peptides, such as, e.g., in the NRPS-generated glycopeptides (Greule et al, 2018).…”

New developments in RiPP discovery, enzymology and engineering

“…18C). 58 They are made from a 27-amino acid precursor peptide encoded in a BGC that also contains a cytochrome P450 (CitB) and a methyltransferase (CitC). A prolyl endopeptidase that removes the leader peptide is encoded outside of the BGC.…”

“…18C). 58 Methylation is a secondary rather than class-dening PTM since the native host produces both cittilin A and a desmethyl congener (cittilin B). The methyltransferase CitC encoded in the BGC was recombinantly expressed and shown to methylate cittilin B indicating it is not leader peptide dependent and likely acts last in the biosynthetic pathway.…”

Biocatalytic synthesis of peptidic natural products and related analogues

“…Many RiPPs classes are featured with sidechain linkages that are catalyzed by different enzyme families ( Arnison et al., 2013 ; Lu et al., 2021 ). For example, a radical S -adenosyl- l -methionine (SAM) enzyme is involved in the formation of Trp-Lys carbon-carbon (C-C) crosslink in streptide ( Schramma et al., 2015 ), whereas a P450 enzyme forms both biaryl C-C and aryl- O -aryl ether crosslinks in the biosynthesis of cittilin A ( Hug et al., 2020 ). In addition, several RiPPs classes are cyclized through the linkages of sidechain-αC of the peptide backbone (e.g., sactipeptides) ( Flühe et al., 2012 ; Himes et al., 2016 ) or include heterocycles (e.g., thiopeptide and linear azol(in)e-containing peptides) ( Burkhart et al., 2017b ; Liao et al., 2009 ).…”