Isolation and Structural Characterization of Capistruin, a Lasso Peptide Predicted from the Genome Sequence of Burkholderia thailandensis E264

Abstract

Lasso peptides are a structurally unique class of bioactive peptides characterized by a knotted arrangement, where the C-terminus threads through an N-terminal macrolactam ring. Although ribosomally synthesized, only the gene cluster for the best studied lasso peptide MccJ25 from Escherichia coli consisting of the precursor protein McjA and the processing and immunity proteins McjB, McjC, and McjD is known. Through genome mining studies, we have identified homologues of all four proteins in Burkholderia thailandensis E264 and predicted this strain to produce a lasso peptide. Here we report the successful isolation of the predicted peptide, named capistruin. Upon optimization of the fermentation conditions, mass spectrometric and NMR structural studies proved capistruin to adopt a novel lasso fold. Heterologous production of the lasso peptide in Escherichia coli showed that the identified genes are sufficient for the biosynthesis of capistruin, which exhibits antimicrobial activity against closely related Burkholderia and Pseudomonas strains. In general, our rational approach should be widely applicable for the isolation of new lasso peptides to explore their high structural stability and diverse biological activity.

Dual substrate‐controlled kinase activity leads to polyphosphorylated lasso peptides

“…3A), in agreement with previous assays of ThcoK with PadeA (13-23) [34]. When ThcoK was incubated with ATP and either PadeA (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) or deletion variant S-PadeA(13-23) DD22, only a singly phosphorylated peptide was significantly produced (Fig. 3B).…”

“…Plasmids were transformed into E. coli BL21(DE3) cells for expression and subsequent purification of Trx fusion proteins, followed by cleavage with TEV protease and further purification as described above for S-ThcoA and S-SyanA. PadeA (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) and PadeA(13-23)-PO 2À 3 were purchased from Biomatik (Cambridge, Ontario, Canada) (> 95% purity).…”

“…3A, ThcoK transferred up to three phosphate groups onto S-ThcoA (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) when ATP served as the phosphate donor; however, a singly phosphorylated peptide appeared as the main product. Conversely, when ATP was replaced with GTP, singly and doubly phosphorylated peptides were produced in nearly equal amounts.…”

“…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].…”

High‐Resolution Crystal Structure of a Lasso Peptide

“…Peptide precursors are ribosomally synthesized and post-translationally modified by proteolytic cleavage and amide bond linkage. [4][5][6][7] Some of the known lasso peptides are secreted into the surrounding media. [8,9] Their proposed biological role is to act as antibacterial agents, presumably to give advantage over competing bacterial species or strains within the same habitat.…”