Lasso Peptides: An Intriguing Class of Bacterial Natural Products

Abstract

Natural products of peptidic origin often represent a rich source of medically relevant compounds. The synthesis of such polypeptides in nature is either initiated by deciphering the genetic code on the ribosome during the translation process or driven by ribosome-independent processes. In the latter case, highly modified bioactive peptides are assembled by multimodular enzymes designated as nonribosomal peptide synthetases (NRPS) that act as a protein-template to generate chemically diverse peptides. On the other hand, the ribosome-dependent strategy, although relying strictly on the 20-22 proteinogenic amino acids, generates structural diversity by extensive post-translational-modification. This strategy seems to be highly distributed in all kingdoms of life. One example for this is the lasso peptides, which are an emerging class of ribosomally assembled and post-translationally modified peptides (RiPPs) from bacteria that were first described in 1991. A wide range of interesting biological activities are known for these compounds, including antimicrobial, enzyme inhibitory, and receptor antagonistic activities. Since 2008, genome mining approaches allowed the targeted isolation and characterization of such molecules and helped to better understand this compound class and their biosynthesis. Their defining structural feature is a macrolactam ring that is threaded by the C-terminal tail and held in position by sterically demanding residues above and below the ring, resulting in a unique topology that is reminiscent of a lariat knot. The ring closure is achieved by an isopeptide bond formed between the N-terminal α-amino group of a glycine, alanine, serine, or cysteine and the carboxylic acid side chain of an aspartate or glutamate, which can be located at positions 7, 8, or 9 of the amino acid sequence. In this Account, we discuss the newest findings about these compounds, their biosynthesis, and their physicochemical properties. This includes the suggested mechanism through which the precursor peptide is enzymatically processed into a mature lasso peptide and crucial residues for enzymatic recognition. Furthermore, we highlight new insights considering the protease and thermal stability of lasso peptides and discuss why seven amino acid residue rings are likely to be the lower limit feasible for this compound class. To elucidate their fascinating three-dimensional structures, NMR spectroscopy is commonly employed. Therefore, the general methodology to elucidate these structures by NMR will be discussed and pitfalls for these approaches are highlighted. In addition, new tools provided by recent investigations to assess and prove the lasso topology without a complete structure elucidation will be summarized. These include techniques like ion mobility-mass spectrometry and a combined approach of thermal and carboxypeptidase treatment with subsequent LC-MS analysis. Nevertheless, even though much was learned about these compounds in recent years, their true native function and the exact enzymatic mechanism of their...

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 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]...…”

“…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.…”

“…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).…”

Dual substrate‐controlled kinase activity leads to polyphosphorylated lasso peptides

“…Extensive post-translational modifications enrich the structural diversity and, thereby, chemical stability and bioactivity of such compounds. Lasso peptides are members of an intriguing family of RiPPs that possess an unusual lariat knot-like structure [6][7][8][9][10][11][12][13][14]: the N terminus of the peptide is covalently cyclized with an acidic side chain to form a 7-, 8-, or 9-residue macrolactam ring; the C terminus is threaded through this ring and trapped by bulky side chains, thus stabilizing the entropically disfavored conformation (Fig. 1A) [9][10][11]13,[15][16][17][18][19][20][21][22][23][24].…”