Cellulonodin-2 and Lihuanodin: Lasso Peptides with an Aspartimide Post-Translational Modification

Abstract

Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded structure. Besides the class-defining isopeptide bond, other post-translational modifications (PTMs) that further tailor lasso peptides have been previously reported. Using genome mining tools, we identified a subset of lasso peptide biosynthetic gene clusters (BGCs) that are colocalized with genes encoding protein l-isoaspartyl methyltransferase (PIMT) homologues. PIMTs have an important role in protein repair, restoring isoaspartate residues formed from asparagine deamidation to aspartate. Here we report a new function for PIMT enzymes in the post-translational modification of lasso peptides. The PIMTs associated with lasso peptide BGCs first methylate an l-aspartate side chain found within the ring of the lasso peptide. The methyl ester is then converted into a stable aspartimide moiety, endowing the lasso peptide ring with rigidity relative to its unmodified counterpart. We describe the heterologous expression and structural characterization of two examples of aspartimide-modified lasso peptides from thermophilic Gram-positive bacteria. The lasso peptide cellulonodin-2 is encoded in the genome of actinobacterium Thermobifida cellulosilytica, while lihuanodin is encoded in the genome of firmicute Lihuaxuella thermophila. Additional genome mining revealed PIMT-containing lasso peptide BGCs in 48 organisms. In addition to heterologous expression, we have reconstituted PIMT-mediated aspartimide formation in vitro, showing that lasso peptide-associated PIMTs transfer methyl groups very rapidly as compared to canonical PIMTs. Furthermore, in stark contrast to other characterized lasso peptide PTMs, the methyltransferase functions only on lassoed substrates.

Bioinformatics-Guided Expansion and Discovery of Graspetides

“…Members of this family are related to IPR000682, which converts l -isoaspartyl and d -aspartyl residues to l -Asp, and IPR027573, which frequently co-occurs with lanthipeptide biosynthetic enzymes . Recently, members of the latter family, OlvS A , TceM, and LihM, were implicated in RiPP biosynthesis , OlvS A catalyzes a three-step reaction (methylation, cyclization, and hydrolysis) of a highly conserved l -Asp to the corresponding l -isoaspartyl residue in OlvA, the cognate lanthipeptide precursor peptide. TceM and LihM introduce stable aspartimide moieties in the lasso peptides, cellulonodin-2 and lihuanodin, respectively.…”

Ribosomally synthesized peptides, foreground players in microbial interactions: recent developments and unanswered questions

“…44,55 Those found in lasso peptides are especially numerous, including glycosylation, phosphorylation, acetylation, C-terminal methylation, β-hydroxylation, deimination, epimerization to d -amino acids, 44 or aspartimide formation from l -aspartate residues, as recently described. 56 Other RiPP classes have also been described as being further modified, such as thiopeptides, and especially lanthipeptides that can accept hydroxylation, epimerization at α-carbons or halogenation, 55 or can acquire β-amino acids with the formation of isoaspartate (iso-Asp) residues. 43 Such additional tailoring PTMs occur due to the dedicated post-tailoring enzymes (see Section 3.1.3).…”

“…They both introduce an aspartimide ring into the macrolactame ring of the lasso peptides. 56 While the conventional role of canonical PIMTs is to reverse iso-Asp residues (the β-amino acids resulting from protein aging) to l -Asp, with aspartimide ( i.e. succinimide) as an intermediate, the lasso peptide O -methyltransferases act directly on Asp residues that are methylated and lead to aspartimide as the final products, and not a reaction intermediate.…”

Unusual Post-Translational Modifications in the Biosynthesis of Lasso Peptides

“…Protein L-isoaspartyl methyltransferases (PIMTs) usually have a crucial role in protein repair, recognizing and repairing abnormal isoaspartate (isoAsp) residues to L-Asp through a SAM-dependent methyl esterification reaction [58]. In total, 48 lasso peptide BGCs were uncovered bearing genes annotated as O-methyltransferases that belong to PIMT homologues, and the extremely conserved Asp6 in all the putative precursor peptides suggested that it might be the modification site [59]. Heterologous expression of two clusters from actinobacterium Thermobifida cellulosilytica (tce) and firmicute Lihuaxuella thermophila (lih) (Figure 9a) resulted in the discovery of cellulonodin-2 and lihuanodin, featuring an unconventional succinimide moiety (also known as aspartimide) in the macrolactam ring.…”

“…Notably, TceM and LihM carried out dehydration on Asp instead of isoAsp, which is in stark contrast to canonical PIMTs. In addition, both TceM and LihM only recognized the threaded lasso peptides rather than linear precursors or isopeptide-bonded rings (Figure 9b) [59].…”