Characterization of Sviceucin from Streptomyces Provides Insight into Enzyme Exchangeability and Disulfide Bond Formation in Lasso Peptides

Abstract

Lasso peptides are bacterial ribosomally synthesized and post-translationally modified peptides. They have sparked increasing interest in peptide-based drug development because of their compact, interlocked structure, which offers superior stability and protein-binding capacity. Disulfide bond-containing lasso peptides are rare and exhibit highly sought-after activities. In an effort to expand the repertoire of such molecules, we heterologously expressed, in Streptomyces coelicolor, the gene cluster encoding sviceucin, a type I lasso peptide with two disulfide bridges originating from Streptomyces sviceus, which allowed it to be fully characterized. Sviceucin and its reduced forms were characterized by mass spectrometry and peptidase digestion. The three-dimensional structure of sviceucin was determined using NMR. Sviceucin displayed antimicrobial activity selectively against Gram-positive bacteria and inhibition of fsr quorum sensing in Enterococcus faecalis. This study adds sviceucin to the type I lasso peptide family as a new representative. Moreover, new clusters encoding disulfide-bond containing lasso peptides from Actinobacteria were identified by genome mining. Genetic and functional analyses revealed that the formation of disulfide bonds in sviceucin does not require a pathway-encoded thiol-disulfide oxidoreductase. Most importantly, we demonstrated the functional exchangeability of the sviceucin and microcin J25 (a non-disulfide-bridged lasso peptide) macrolactam synthetases in vitro, highlighting the potential of hybrid lasso synthetases in lasso peptide engineering.

A new genome-mining tool redefines the lasso peptide biosynthetic landscape

“…This suggests some will display more esoteric activities or influence signal transduction. This last possibility is consistent with sviceucin’s inhibition of Enterococcus faecalis quorum sensing 46 . One such lasso peptide described herein that lacks bacterial growth-suppressive activity, citrulassin A, belongs to a 55-membered family.…”

“…All lasso peptides were thermally stable, showing no unthreading by HPLC after extended heating, and were either resistant or impervious to carboxypeptidase Y digestion (Supplementary Fig. 14) 46, 47 . These data strongly support a threaded lasso topology.…”

New Insights into the Biosynthetic Logic of Ribosomally Synthesized and Post-translationally Modified Peptide Natural Products

“…Mutational studies using the sactipeptide RiPP biosynthetic machinery has also been performed, revealing the importance of the RRE for sactipeptide maturation (Wieckowski et al, 2015). Interestingly, the RRE domain sometimes may exist as a stand-alone protein and not as a subdomain within a RiPP biosynthetic enzyme as recently observed for lasso peptides and thiazole/oxazole-modified peptide gene clusters (Burkhart et al, 2015, Dunbar et al, 2015, Elsayed et al, 2015, Gavrish et al, 2014, Inokoshi et al, 2012, Li et al, 2015, Metelev et al, 2015). …”

Insights into the Unique Phosphorylation of the Lasso Peptide Paeninodin

“…Sequences of representative C proteins accessible from published functional clusters (6,7,10,11,18,(22)(23)(24)(25), in addition to those of the six C proteins from kinase-containing clusters, were used as input. The phylogenetic analysis shows clear separation of gene clusters into five clades, coinciding with the bacterial phylum of origin as well as the organization of genes within the clusters (Fig.…”