Dissecting the Maturation Steps of the Lasso Peptide Microcin J25 in vitro

Abstract

Microcin J25 is the archetype of a growing class of bacterial ribosomal peptides possessing a knotted topology (lasso peptides). It consists of an eight‐residue macrolactam ring through which the C‐terminal tail is threaded. It is biosynthesized as a precursor that is processed by two maturation enzymes (McjB/McjC). Insights into the mechanism of microcin J25 biosynthesis have been provided previously by mutagenesis of the precursor peptide in vivo. In this study we have demonstrated distinct functions of McjB and McjC in vitro for the first time, based on the detection of reaction intermediates. McjB was characterized as a new ATP‐dependent cysteine protease, whereas McjC was confirmed to be a lactam synthetase. The two enzymes were functionally interdependent, likely forming a structural complex. Their substrate preference was directly investigated with the aid of mutated precursor peptides. Depending on the substitutions, microcin J25 variants with either a lasso or branched‐cyclic topology could be generated in vitro.

Dual substrate‐controlled kinase activity leads to polyphosphorylated lasso peptides

“…Generally, four primers were used to generate two PCR products (one with P1 and P4; the other with P2 and P3), which were digested with DpnI and combined for further treatment according to the SLIM protocol. Plasmids pET48b-ThcoA(12-22), pET48b-ThcoA(12-22) insD22, and pET48b-SyanA(14-24) were prepared from templates pET48b-ThcoA, pET48b-ThcoA (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), and pET48b-SyanA, respectively (see Table S2 for primers). To generate pET48b-PadeA(13-23)D22D, two rounds of mutagenesis were performed: pET48b-PadeA [34] served as a template to produce pET48b-PadeA(13-23), which then served as a template to produce pET48b-PadeA(13-23) D22D (see Table S2 for primers).…”

“…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).…”

“…We sought to test whether subtle alterations in sequence could modulate the observed polyphosphorylation activity. Thus, we prepared S-ThcoA (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), S-ThcoA(12-22)insD22 and S-PadeA(13-23)DD22 from Trx fusions and purchased synthetic PadeA (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Each peptide was then tested with ThcoK, which displayed significantly higher activity than SyanK.…”

“…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].…”

“…The only known examples include C-terminal methylation of lassomycin and Trp side chain hydroxylation of some RES-701 family lasso peptides [7,9,11,12,19,21,[25][26][27][28][29][30][31][32][33]. Recently, when characterizing paeninodin, a newly discovered lasso peptide from firmicutes, we identified several cryptic clades of gene clusters that feature a lasso peptide-tailoring kinase (Fig.…”