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 processed 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-terminal sequence threaded through the macrocycle. This unique lariat topology, which typically provides considerable stability toward 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 Arg deimination to yield citrulline (Cit). Although a Cit-containing lasso peptide (i.e., citrulassin) was serendipitously discovered during a genome-guided campaign, the gene(s) responsible for Arg deimination has remained unknown. Herein, we describe the use of reactivity-based screening to discriminate bacterial strains that produce Arg- versus Cit-bearing citrulassins, yielding 13 new lasso peptide variants. Partial phylogenetic profiling identified a distally encoded peptidyl arginine deiminase (PAD) gene ubiquitous to the Cit-containing variants. Absence of this gene correlated strongly with lasso peptide variants only containing Arg (i.e., des-citrulassin). Heterologous expression of the PAD gene in a des-citrulassin producer resulted in the production of the deiminated analog, confirming PAD involvement in Arg deimination. The PADs were then bioinformatically surveyed to provide a deeper understanding of their taxonomic distribution and genomic contexts and to facilitate future studies that will evaluate any additional biochemical roles for the superfamily.

Recent Advances and Perspectives on Expanding the Chemical Diversity of Lasso Peptides

“…Interestingly, two distinct Streptomyces species (S. avermitilis and S. torulosus) were found to produce citrulassin variants with identical core peptide sequences, but with the citrulline modification absent in one and present in the other. Comparison of genomic sequences enabled the identification of a potentially responsible peptidylarginine deiminase (PAD), which, unlike other lasso peptide-tailoring enzymes, is not encoded locally in the lasso peptide gene cluster (Harris et al, 2020). In vivo complementation experiments confirmed the participation of the PAD (WP_064069847.1) in the biosynthesis of deiminated lasso peptides (Figure 6A).…”

“…Nuclear magnetic resonance (NMR) and tandem mass spectrometry (MS/MS) analyses revealed that Arg9 of the lasso peptide was converted to citrulline. To determine the identity of the enzyme responsible for Arg deimination, researchers performed reactivity-based screening with a selective probe for citrulline-containing natural products, leading to the discovery of 13 citrulassin variants (Harris et al, 2020). Interestingly, two distinct Streptomyces species (S. avermitilis and S. torulosus) were found to produce citrulassin variants with identical core peptide sequences, but with the citrulline modification absent in one and present in the other.…”

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

“…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 has revealed tailoring enzymes that subject lasso peptides to further post-translational modifications (PTMs). Modifications that have been confirmed experimentally include disulfide bonds, , C-terminal methylation, , acetylation, citrullination, phosphorylation, glycosylation, epimerization, , and β-hydroxylation. , 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. , …”

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 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 -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.…”