Class V Lanthipeptide Cyclase Directs the Biosynthesis of a Stapled Peptide Natural Product

Abstract

Lanthipeptides are a class of cyclic peptides characterized by the presence of one or more lanthionine (Lan) or methyllanthionine (MeLan) thioether rings. These cross-links are produced by α,β-unsaturation of Ser or Thr residues in peptide substrates by dehydration, followed by a Michael-type conjugate addition of Cys residues onto the dehydroamino acids. Lanthipeptides may be broadly classified into at least five different classes, and the biosynthesis of classes I–IV lanthipeptides requires catalysis by LanC cyclases that control both the site-specificity and the stereochemistry of the conjugate addition. In contrast, there are no current examples of LanCs that occur in class V biosynthetic clusters, despite the presence of lanthionine rings in these compounds. In this work, bioinformatics-guided co-occurrence analysis identifies more than 240 putative class V lanthipeptide clusters that contain a LanC cyclase. Reconstitution studies demonstrate that the cyclase-catalyzed product is notably distinct from the product formed spontaneously. Stereochemical analysis shows that the cyclase diverts the final product to a configuration that is distinct from one that is energetically favored. Structural characterization of the final product by multi-dimensional NMR spectroscopy reveals that it forms a helical stapled peptide. Mutational analysis identified a plausible order for cyclization and suggests that enzymatic rerouting to the final structure is largely directed by the construction of the first lanthionine ring. These studies show that lanthipeptide cyclases are needed for the biosynthesis of some constrained peptides, the formations of which would otherwise be energetically unfavored.

Refactoring and Heterologous Expression of Class III Lanthipeptide Biosynthetic Gene Clusters Lead to the Discovery of N,N-Dimethylated Lantibiotics from Firmicutes

“…Lanthipeptides are the largest subfamily of ribosomally synthesized and post-translationally modified peptides (RiPPs). These peptide natural products bear characteristic intramolecular lanthionine and/or labionin rings and are often referred to as lantibiotics due to their prevalent antimicrobial properties. , They are subdivided into five classes depending on their biosynthetic machinery. ,− Class III lanthipeptides are an emerging class, beginning with the report of their first member, labyrinthopeptins, in 2010. , Consistent with the literature, , our present bioinformatic analysis of 223,243 bacterial and archaeal genomes revealed that class III lanthipeptide biosynthetic gene clusters (BGCs) are widely distributed in bacterial phyla, with Actinobacteria being the most abundant and Firmicutes being the second most abundant harboring these BGCs. Compared to Actinobacteria, a known major source for class III lanthipeptide discovery, − no class III lanthipeptide structures had been reported from Firmicutes when we initiated the present study, and now only andalusicin, amylopeptin, bacinapeptin, and paenithopeptin have been reported, still leaving class III lanthipeptides in Firmicutes significantly underexplored.…”

syn-Elimination of glutamylated threonine in lanthipeptide biosynthesis

“…S4, ESI†). The observed NOEs differ significantly from what is observed for ( Z )-Dhb residues in previously characterized lanthipeptides 30,31 (Fig. S5 and S6, ESI†) and linaridins.…”

Deciphering the Biosynthesis of Novel Class I Lanthipeptides from Marine Pseudoalteromonas Reveals a Dehydratase PsfB with Dethiolation Activity

“…Recently, the class V lanthipeptide biosynthetic pathway was identified and characterized. A kinase/lyase pair (LanK/LanY) and a class I cyclase LanC were found to be responsible for the dehydration and cyclization reactions. ,− Apart from the difference between the biosynthetic enzymes, five classes of lanthipeptides can also be distinguished based on the biosynthetic machinery utilized by these enzymes. Class I LanB dehydratase uses glutamyl-tRNA Glu to activate the hydroxyl side chains of Ser/Thr residues followed by glutamate elimination , (Figure A).…”