Bioinformatic Mapping of Radical S-Adenosylmethionine-Dependent Ribosomally Synthesized and Post-Translationally Modified Peptides Identifies New Cα, Cβ, and Cγ-Linked Thioether-Containing Peptides - PubMed (original) (raw)

Bioinformatic Mapping of Radical S-Adenosylmethionine-Dependent Ribosomally Synthesized and Post-Translationally Modified Peptides Identifies New Cα, Cβ, and Cγ-Linked Thioether-Containing Peptides

Graham A Hudson et al. J Am Chem Soc. 2019.

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Abstract

Recently developed bioinformatic tools have bolstered the discovery of ribosomally synthesized and post-translationally modified peptides (RiPPs). Using an improved version of Rapid ORF Description and Evaluation Online (RODEO 2.0), a biosynthetic gene cluster mining algorithm, we bioinformatically mapped the sactipeptide RiPP class via the radical S-adenosylmethionine (SAM) enzymes that form the characteristic sactionine (sulfur-to-α carbon) cross-links between cysteine and acceptor residues. Hundreds of new sactipeptide biosynthetic gene clusters were uncovered, and a novel sactipeptide "huazacin" with growth-suppressive activity against Listeria monocytogenes was characterized. Bioinformatic analysis further suggested that a group of sactipeptide-like peptides heretofore referred to as six cysteines in forty-five residues (SCIFFs) might not be sactipeptides as previously thought. Indeed, the bioinformatically identified SCIFF peptide "freyrasin" was demonstrated to contain six thioethers linking the β carbons of six aspartate residues. Another SCIFF, thermocellin, was shown to contain a thioether cross-linked to the γ carbon of threonine. SCIFFs feature a different paradigm of non-α carbon thioether linkages, and they are exclusively formed by radical SAM enzymes, as opposed to the polar chemistry employed during lanthipeptide biosynthesis. Therefore, we propose the renaming of the SCIFF family as radical non-α thioether peptides (ranthipeptides) to better distinguish them from the sactipeptide and lanthipeptide RiPP classes.

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Figures

Figure 1.

Figure 1.. Sactipeptide biosynthesis and ring topologies.

(A) Schematic overview of sactipeptide biosynthesis. (B) Proposed mechanism for the rSAM-dependent formation of the sactipeptide S–Cα thioether crosslink. (C) Precursor peptide sequence and ring topologies of characterized sactipeptides (blue, S–Cα linkage; yellow, S-S linkage; black, uncharacterized). The requisite rSAM enzyme name is given in parentheses. Characterization of the six Cys in forty-five residues (SCIFFs) is limited to in vitro enzymatic assay for which only one thioether linkage was observed. Data shown later in this manuscript support a Cys-Thr linkage different to what has been previously reported and further that SCIFFs are not sactipeptides. * Hyicin 4244 has not been structurally characterized, thus the linkages indicated are speculative.

Figure 2.

Figure 2.. Huazacin from Bacillus thuringiensis serovar huazhongensis.

(A) Biosynthetic gene cluster. The sactipeptide synthases HuaB and HuaC (NCBI accession identifiers: EEM79976.1 and EEM79977.1, respectively) feature an N-terminal RRE domain and C-terminal SPASM domain similar to other sactisynthases., (B) Precursor peptide sequence and chemical structure of huazacin. The hua gene cluster contains two identical copies of the precursor peptide shown.

Figure 3.

Figure 3.. Sequence similarity network of rSAM enzymes.

rSAM enzymes involved in sactipeptide and SCIFF biosynthesis were used as BLAST inputs to gather additional rSAM enzymes predicted to form RiPP thioether linkages (n = 4,693). Sequences were analyzed by EFI-EST and visualized using Cytoscape. Nodes are colored based on the category of its associated precursor peptide (see legend). Sequences sharing >70% identity are conflated as a single node, and connected nodes indicate a similarity score of < 1e-50.,, Sequence logos for core peptides associated with these rSAMs are shown in Figures S1 and S9. QhpD, non-RiPP rSAM involved in modifying quinohemoprotein amine dehydrogenase.

Figure 4.

Figure 4.. Freyrasin biosynthetic gene cluster and related peptides.

(A) Biosynthetic gene cluster diagram. (B) Freyrasin precursor peptide sequence with six CX3D motifs and thioether topologies highlighted. (C) Alignment of additional freyrasin members. Leader peptide residues are negatively numbered relative to the core peptide, which we predict begins with Gly.

Figure 5.

Figure 5.. Freyrasin contains beta-linked thioethers.

(A) Cys-Asp (S–Cβ) connectivity with hydrogens colored pink (Cys) or blue (Asp). (B) 1H-1H NOESY correlations confirming a S–Cβ thioether linkage between Cys26-Asp30. (C) Same as panel B but for Cys20-Asp24.

Figure 6.

Figure 6.. Thermocellin contains a Cys-Thr (S-Cγ) thioether.

(A) Coexpression of the thermocellin precursor containing labeled Thr (blue) features a −2 Da shift relative to unmodified peptide, demonstrating a S-Cγ linkage was formed. The modified peptide was treated with endoproteinase GluC and unmodified Cys were alkylated with _N_-ethylmaleimide (red) Residues are numbered based on the sequence after GluC treatment. (B) Structure of the isotopically labeled thioether linkage. (C) MS/MS analysis yielded fragmentation consistent with a macrocycle formed between Cys9 and Thr11. Error tables for assigned ions are available in Table S6.

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