Reactivity-based screening for citrulline-containing natural products reveals a family of bacterial peptidyl arginine deiminases

Abstract

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a family of natural products defined by a genetically encoded precursor peptide that is tailored by associated biosynthetic enzymes to form the mature product. Lasso peptides are a class of RiPP defined by an isopeptide linkage between the N-terminal amine and an internal Asp/Glu residue with the C-terminus threaded through the macrocycle. This unique lariat topology, which provides considerable stability towards heat and proteases, has stimulated interest in lasso peptides as potential therapeutics. Post-translational modifications beyond the class-defining, threaded macrolactam have been reported, including one example of arginine deimination to yield citrulline. Although a citrulline-containing lasso peptide (i.e., citrulassin) was serendipitously discovered during a genome-guided campaign, the gene(s) responsible for arginine deimination has remained unknown. Herein we describe the use of reactivity-based screening to discriminate bacteria that produce arginine-versus citrulline-bearing citrulassins, culminating in the discovery and characterization of 11 new lasso peptide variants. Phylogenetic profiling identified a distally encoded peptidyl arginine deiminase (PAD) gene ubiquitous to the citrulline-containing variants. Absence of this gene correlated strongly with citrulassin variants only containing arginine (des-citrulassin). Heterologous expression of the PAD in a non-citrulassin producer resulted in the production of the deiminated analog, confirming PAD involvement in arginine deimination. The family of PADs were then bioinformatically surveyed for a deeper understanding of its genomic context and potential role in post-translational modification of RiPPs.

Cellulonodin-2 and Lihuanodin: Lasso Peptides with an Aspartimide Post-Translational Modification

“…3,17,24,25 Beyond the minimal BGC required for lasso peptide biosynthesis and commonly associated transporters and isopeptidases, genome mining of the ever-growing database of bacterial genome sequences 12 has revealed tailoring enzymes that subject lasso peptides to further post-translational modifications (PTMs). Modifications that have been confirmed experimentally include disulfide bonds, 26,27 C-terminal methylation, 19,28 acetylation, 29 citrullination, 30 phosphorylation, 31 glycosylation, 32 epimerization, 26,33 and β-hydroxylation. 6,34 All previously described lasso peptide PTMs occur in either the loop or tail regions of the lasso peptide, and these auxiliary enzymes recognize the linear precursor rather than the matured lasso as a substrate.…”

Sterol Sponge Mechanism Is Conserved for Glycosylated Polyene Macrolides

“…This chemistry-based approach can be guided by genomic prioritization to only include strains capable of installing the targeted functional group, thus expediting natural product discovery. We have used reactivity-based screening to identify α/β-unsaturated carbonyl-containing natural products via 1,4-nucleophilic addition, reactive carbonyl-containing compounds via oxime ligation, and primary ureido-group-containing compounds via nucleophilic addition onto glyoxal-based probes. − Other research groups have reported related approaches for elucidating the proteomic targets of natural products, chemoselective natural product enrichment, and methodology to covalently target di/trienes using a nitrosopyridine-based probe. − To target the electron-rich polyene common to all GPMs (Supplemental Figure S1), we co-opted the well-known tetrazine ligation that was recently employed to detect isonitrile-containing natural products. , …”