Structure and biosynthesis of a macrocyclic peptide containing an unprecedented lysine-to-tryptophan crosslink

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. Herein we have combined tools from natural products discovery and mechanistic enzymology to report 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.

Characterization of the macrocyclase involved in the biosynthesis of RiPP cyclic peptides in plants

“…As presegetalin A1 [27][28][29][30][31][32] binding induces closure of the PCY1 structure, addition of exogenous peptide would be expected to inhibit productive turnover of the presegetalin A1 [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] substrate. We tested this hypothesis using a 1-h end-point reaction of PCY1 incubated with 30 μM of substrate in the presence of stoichiometric and 10-fold excess of the presegetalin A1 [20][21][22][23][24][25][26][27][28][29][30][31][32] linker+follower peptide product, as well as 100-fold excess of the presegetalin A1 [27][28][29][30][31][32] follower peptide (SI Appendix, Fig.…”

“…We then tested whether PCY1 could generate a macrocyclic product using a nonproteinogenic amine. To this end, we used a presegetalin A1 [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] substrate that contained an N-terminal aminohexanoic acid (Ahx) in place of Gly14. PCY1 largely produced a linear product with this substrate, although a minor amount of cyclic [AhxVPVWA] could also be detected (SI Appendix, Table S3 and Fig.…”

“…We next interrogated the extent to which the enzyme could accommodate primary amines other than a peptide α-amino group for transamidation. First, we used a variant presegetalin A1 [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] substrate peptide that contained both the α-amino terminus and a side-chain amine (Val15→Lys). Kinetic characterization of PCY1 using the Val15→Lys presegetalin A1 [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] as a substrate demonstrates that the K M increased by 10-fold relative to the cognate presegetalin A1 [14][15][16][17][18][19][20][21][22]...…”

“…Recent studies of various RiPP biosynthetic enzymes have provided several examples of biocatalysts that can generate a macrocyclic product from a linear peptide substrate (14,28,30,31,(52)(53)(54). Such enzymes represent a next-generation technological utility, but many of these catalysts are often slow or require sequence constraints on substrates.…”

“…RiPPs are translated by the ribosome as linear peptides that are subsequently enzymatically modified to yield compounds with a range of biological activities (10). Many RiPPs undergo enzymatic macrocyclization, as observed in several diverse classes of compounds including bottromycins (11), cyanobactins (12), lanthipeptides (13), streptides (14), lasso peptides (15), and thiopeptides (16). Understanding the biosynthetic pathways for RiPP macrocyclization continues to be important for biotechnological application, for the identification of new gene clusters, and for the production of novel RiPP derivatives (17).…”