Structure and biosynthesis of a macrocyclic peptide containing an unprecedented lysine-to-tryptophan crosslink - PubMed (original) (raw)

Kelsey R Schramma et al. Nat Chem. 2015 May.

Abstract

Streptococcal bacteria use peptide signals as a means of intraspecies communication. These peptides can contain unusual post-translational modifications, providing opportunities for expanding our understanding of nature's chemical and biosynthetic repertoires. Here, we have combined tools from natural products discovery and mechanistic enzymology to elucidate the structure and biosynthesis of streptide, a streptococcal macrocyclic peptide. We show that streptide bears an unprecedented post-translational modification involving a covalent linkage between two unactivated carbons within the side chains of lysine and tryptophan. The biosynthesis of streptide was addressed by genetic and biochemical studies. The former implicated a new SPASM-domain-containing radical SAM enzyme StrB, while the latter revealed that StrB contains two [4Fe-4S] clusters and installs the unusual lysine-to-tryptophan crosslink in a single step. By intramolecularly stitching together the side chains of lysine and tryptophan, StrB provides a new route for biosynthesizing macrocyclic peptides.

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Figures

Figure 1

Figure 1. Biosynthetic gene cluster and structure of streptide

a, The str cluster consists of a precursor peptide (strA), a radical SAM gene (strB), and a putative transporter (strC). It is activated by the shp/rgg quorum sensing system, which encodes a peptide hormone (shp) and a transcriptional regulator (rgg)14. b, Structural elucidation of streptide by NMR. Key correlations used to solve the structure are indicated. c, Structural and stereochemical assignment of streptide. d, Computational model of the three-dimensional structure of streptide calculated using CYANA and NMR NOESY constraints. e, HR-HPLC-MS analysis of the extracts of wt S. thermophilus (red trace) and a strB::ery mutant (black trace). Shown is the extracted ion intensity for streptide (m/z 989.4879). The starred peak corresponds to streptide.

Figure 2

Figure 2. Proposed biosynthesis for streptide

StrA encodes a 30mer substrate. In our model, StrB installs the Lys-to-Trp crosslink, while StrC, possibly in concert with other protease(s), secretes the mature 9mer streptide. A 9mer, 20mer, and the full-length 30mer StrA, synthesized in this study, are shown. The 9mer sequence of streptide is shown in bold; the K and W residues involved in the cyclization are shown in red.

Figure 3

Figure 3. StrB contains two [4Fe-4S] clusters

a, Alignment of the SPASM motif of StrB with that of anSME. Cys residues that bind the AuxI and AuxII clusters (in anSME) are shown in red and blue, respectively. StrB lacks a key Cys ligand to the AuxI cluster. b, UV-vis spectrum of reconstituted StrB showing a 320 nm shoulder and a 395 nm feature, characteristic of [4Fe-4S] clusters. c, X-band CW EPR spectrum of reconstituted StrB yielding gx, gy, and gz of 1.86, 1.94, and 2.05, respectively. d, EPR spectrum of C409A/C415A-StrB with gII and g of 2.06 and 1.93 typical for the SAM-cleaving [4Fe-4S]+ cluster.

Figure 4

Figure 4. StrB catalyzes Lys-to-Trp crosslink formation in StrA

a, Qtof-HPLC-MS assay for 30mer product formation. Activity assays were carried out in the absence of 30mer substrate (gray trace), reductant (black trace), SAM (blue trace), StrB (green trace), or containing all components (red trace). The 30mer substrate (S) and product (P) peaks are marked. The traces have been offset in both axes for clarity. Inset, HR-ESI-MS spectrum of product 30mer ([M+2H]2+ calc 1656.29144). b, Overlaid TOCSY NMR spectra of StrA (blue) and the product 30mer (red). The substrate contains two unmodified Trp residues (WC and WN), while the product spectrum reveals a C-terminal Trp (WC) and the modified Trp lacking a 1H at the indole-C7 (Wm). The peaks have been assigned in the overlaid 1H traces. See Supplementary Fig. 15 for the upstream region. c, HR-MS/MS spectrum of product 30mer. The observed b and y ions are shown in the inset. Fragmentation at all peptide bonds is observed except for those in the KGDGW sequence, consistent with macrocyclization within this motif (see Supplementary Table 5).

Figure 5

Figure 5. Mechanistic model for Lys-to-Trp crosslink formation catalysed by StrB

The FeS clusters in red and blue correspond to the SAM-cleaving active site cluster and the auxiliary cluster, respectively. Reductive activation of SAM leads to formation of 5′-dA•, which abstracts a Lys β-hydrogen. The radical thus formed reacts with the indole side chain to create the Lys-to-Trp crosslink and an indolyl radical. Deprotonation and rearomatization with concomitant reduction of the auxiliary Fe-S cluster completes the synthesis of crosslinked StrA.

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