Microbial natural products comprise diverse architectures that are generated by equally diverse biosynthetic strategies. In peptide natural products, amino acid sidechains are frequently used as sites of modification to generate macrocyclic motifs. Backbone amide groups, among the most stable of biological moieties, are rarely used for this purpose. Here we report the discovery and biosynthesis of bicyclostreptins—peptide natural products from Streptococcus spp. with an unprecedented structural motif consisting of a macrocyclic β-ether and a heterocyclic sp3–sp3 linkage between a backbone amide nitrogen and an adjacent α-carbon. Both reactions are installed, in that order, by two radical S-adenosylmethionine (RaS) metalloenzymes. Bicyclostreptins are produced at nM concentrations and are potent growth regulation agents in Streptococcus thermophilus. Our results add a distinct and unusual chemotype to the growing family of ribosomal peptide natural products, expand the already impressive catalytic scope of RaS enzymes, and provide avenues for further biological studies in human-associated streptococci.
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Plasmids and strains used in this study are described in Supplementary Tables 1 and 2. All oligonucleotides are shown in Supplementary Table 3. The sequences of codon-optimized gene fragments are provided in Supplementary Note 1. Other relevant data supporting the findings of this study are available within the paper and the supplementary material. Raw NMR data used to elucidate natural product structures are available from the corresponding author upon reasonable request.
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We thank B. A. Johnson for technical assistance with isolation of bicyclostreptin A as well as the Eli Lilly Edward C. Taylor Fellowship in Chemistry (to K.A.C.) and the National Science Foundation (NSF GRFP to L.B.B. and NSF CAREER Award to M.R.S.) for financial support.
The authors declare no competing interests.
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(a) All y-ions N-terminal to His34 and b-ions C-terminal to His34 for the HghCD product are -2 Da relative to substrate. The same y-ions from the HghBCD product are -4 Da, while b40 remains 2 Da lighter. This pattern points to modifications at His34 within the ‘HGH’ motif. (b) Trypsinolysis of the HghBD or HghBCD products gives the fragments shown. The C-terminal 8mer captures both -2 Da (HghBD) and -4 Da (HghBCD) products. Cleavage does not occur at the Lys residue in red.
(a) Reaction of HghC or HghB with SAM in the absence of HghA. Shown are HR-MS extracted ion chromatograms for 5’-dA after an overnight reaction (18 h). The cleaved 5ꞌ-dA product is seen with both reductants but not in the absence of enzyme. (b) Detection of 5ꞌ-dA after the reaction of HghC or HghB with substrate and SAM after 18 h. Note, different elution programs were carried out in panels a and b, thus explaining the different retention times for 5’-dA (see Methods).
Extended Data Fig. 3 Time-dependent formation of the HghC product as measured by HPLC-Qtof-MS extracted ion chromatography.
The average of three independent samples are shown; bars represent standard error.
Extended Data Fig. 4 Sequence alignment of HghD from S. agalactiae or S. thermophilus with PqqD and WgkC.
No substantial homology is observed.
Extended Data Fig. 5 Biosynthetic pathway of bicyclostreptins A and B from S. thermophilus JIM 8232.
Blue spheres represent unmodified amino acids in the core region with the indicated one-letter code. Gray spheres are amino acids in the leader sequence.
Extended Data Fig. 6 Summary of growth inhibition assays of bicyclostreptins A and C and the variants shown against select bacteria.
Shown are MICs in μM (black and red) or IC50 values in μM (blue), which were calculated using the averages of two independent biological samples.
The averages of two biologically independent samples are shown.
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Bushin, L.B., Covington, B.C., Clark, K.A. et al. Bicyclostreptins are radical SAM enzyme-modified peptides with unique cyclization motifs. Nat Chem Biol 18, 1135–1143 (2022). https://doi.org/10.1038/s41589-022-01090-8