The radical SAM superfamily (RSS), arguably the most functionally diverse enzyme superfamily, is also one of the largest with ∼700 K members currently in the UniProt database. The vast majority of the members have uncharacterized enzymatic activities and metabolic functions. In this Perspective, we describe RadicalSAM.org, a new web-based resource that enables a user-friendly genomic enzymology strategy to explore sequence-function space in the RSS. The resource attempts to enable identification of isofunctional groups of radical SAM enzymes using sequence similarity networks (SSNs) and the genome context of the bacterial, archaeal, and fungal members provided by genome neighborhood diagrams (GNDs). Enzymatic activities and in vivo functions frequently can be inferred from genome context given the tendency for genes of related function to be clustered. We invite the scientific community to use RadicalSAM.org to (i) guide their experimental studies to discover new enzymatic activities and metabolic functions, (ii) contribute experimentally verified annotations to RadicalSAM.org to enhance the ability to predict novel activities and functions, and (iii) provide suggestions for improving this resource.
The radical non-α-carbon thioether peptides (ranthipeptides) are a newly described class of ribosomally synthesized and post-translationally modified peptide (RiPP). Ranthipeptide biosynthetic gene clusters are characterized by a Cys-rich precursor peptide and a radical Sadenosylmethionine (rSAM)-dependent enzyme that forms a thioether linkage between a Cys donor and an acceptor residue. Unlike the sulfur-to-α-carbon linked thioether peptides (sactipeptides), known ranthipeptides contain thioethers to either the βor γ-carbon (i.e. non-αcarbon) of an acceptor residue. Recently, we reported the discovery of freyrasin, a ranthipeptide from Paenibacillus polymyxa, which contains six thioethers from Cys-X 3 -Asp motifs present in the precursor peptide (PapA). The linkages are exclusively to the β-carbon of Asp (S-Cβ). In this report, we performed mutational analysis of PapA and the cognate thioether-forming rSAM enzyme (PapB) to define the substrate scope. Using a mass spectrometry-based activity assay, our data show that PapB is intolerant towards Ala and Asn in the acceptor position but will tolerate Glu-containing variants. NMR spectroscopic data of a Glu variant demonstrated that the thioether linkage was to the 4-position of Glu (S-Cγ). Furthermore, we demonstrate that PapB is intolerant to expansion and contraction of the thioether motifs (Cys-X n -Asp, n=2,4), although a minimal substrate featuring only one Cys-X 3 -Asp motif was competent for thioether formation. Akin to the sactipeptides, PapB was dependent on a RiPP recognition element (RRE) to bind the cognate precursor peptide, with deletion resulting in loss-of-function in vivo. The activity of PapB could be restored in vivo by supplying the excised RRE in trans. Finally, we reconstituted the activity of PapB in vitro, which lead to modification of all six Cys residues in PapA. These studies provide insights into ranthipeptide biosynthesis and expand our understanding of rSAM enzyme chemistry in natural product biosynthesis.
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a promising source of new antimicrobials in the face of rising antibiotic resistance. Here, we report a scalable platform that combines high-throughput bioinformatics with automated biosynthetic gene cluster refactoring for rapid evaluation of uncharacterized gene clusters. As a proof of concept, 96 RiPP gene clusters that originate from diverse bacterial phyla involving 383 biosynthetic genes are refactored in a high-throughput manner using a biological foundry with a success rate of 86%. Heterologous expression of all successfully refactored gene clusters in Escherichia coli enables the discovery of 30 compounds covering six RiPP classes: lanthipeptides, lasso peptides, graspetides, glycocins, linear azol(in)e-containing peptides, and thioamitides. A subset of the discovered lanthipeptides exhibit antibiotic activity, with one class II lanthipeptide showing low µM activity against Klebsiella pneumoniae, an ESKAPE pathogen. Overall, this work provides a robust platform for rapidly discovering RiPPs.
The radical S-adenosylmethionine (rSAM) superfamily has become a wellspring for discovering new enzyme chemistry, especially regarding ribosomally synthesized and post-translationally modified peptides (RiPPs). Here, we report a compendium of nearly 15,000 rSAM proteins with high-confidence involvement in RiPP biosynthesis. While recent bioinformatics advances have unveiled the broad sequence space covered by rSAM proteins, the significant challenge of functional annotation remains unsolved. Through a combination of sequence analysis and protein structural predictions, we identified a set of catalytic site proximity residues with functional predictive power, especially among the diverse rSAM proteins that form sulfur-to-α carbon thioether (sactionine) linkages. As a case study, we report that an rSAM protein from Streptomyces sparsogenes (StsB) shares higher full-length similarity with MftC (mycofactocin biosynthesis) than any other characterized enzyme. However, a comparative analysis of StsB to known rSAM proteins using "catalytic site proximity" predicted that StsB would be distinct from MftC and instead form sactionine bonds. The prediction was confirmed by mass spectrometry, targeted mutagenesis, and chemical degradation. We further used "catalytic site proximity" analysis to identify six new sactipeptide groups undetectable by traditional genome-mining strategies. Additional catalytic site proximity profiling of cyclophane-forming rSAM proteins suggests that this approach will be more broadly applicable and enhance, if not outright correct, protein functional predictions based on traditional genomic enzymology principles.
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