Investigation of the Novel PTM Bearing Lasso Peptide Cellulonodin-2 and the Enzyme that Modifies it

Abstract

Lasso peptides, a class of ribosomally synthesized and post-translationally modified peptides (RiPPs), represent an abounding source of compounds which have become popular subjects of study for their [1]rotaxane structure and diverse antimicrobial activity. Genome mining has introduced an added level of attention to this field by identifying interesting, putative lasso biosynthetic gene clusters (BGCs) including a subset of lasso peptides which display further post-translational modifications (PTMs) beyond their characteristic isopeptide bond. In this thesis, heterologous expression in E. coli was used to produce cellulonodin-2, a lasso peptide from Thermobifida cellulosilytica which displays a novel PTM on its 6th residue, making it the first lasso peptide with a PTM in its ring portion. Mass spectrometry and single-residue mutagenesis revealed that this modification is the conversion of an aspartate residue to a succinimide. The results of in vitro reactions with purified TceMT, the protein-isoaspartyl-methyltransferase (PiMT) homologue which modifies cellulonodin-2, provided the first experimental evidence for a PTM introduced to a mature lasso peptide. Furthermore, TceMT was shown to modify specifically the threaded lasso topology but showed some promiscuity with regard to amino acid sequence. This study of cellulonodin-2 and TceMT challenges the accepted notions that succinimides are unstable moieties and that Gram-positive bacteria lack PiMTs.