The Supersized Class III Lanthipeptide Stackepeptin Displays Motif Multiplication in the Core Peptide.

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides bearing the characteristic amino acids lanthionine and/or labionin. Here, we report on the discovery and characterization of the stackepeptins, produced by the Actinomycete Stackebrandtia nassauensis DSM-44728(T). The stackepeptins are the first supersized class III lanthipeptides to be discovered. Unlike other class III lanthipeptides, they consist of three lanthionine/labionin moieties instead of two. In this study, both in vivo and in vitro maturation of the peptides have been investigated. Studies involving the wild type strain showed culture medium-dependent production of three stackepeptins consisting of one common N-terminal labionin ring and varying dehydration and cyclization patterns in the C-terminal rings. On the other hand, in vitro assessment of the heterologously expressed modifying enzyme StaKC, yielded one major product with an N-terminal lanthionine and C-terminal labionins. The discrepancy between in vivo and in vitro processing was discovered to be sequence-dependent and also implies that in vivo processing is facilitated by additional factors in the cell. Furthermore, a Ser → Ala scan revealed the importance of C-terminal ring formation for full in vitro maturation of the stackepeptins. StaKC showed promiscuity toward the phosphorylating cosubstrate with a significant preference for purine nucleotides. Finally, in contrast to other known class III lanthipeptides, in vitro experiments showed that the leader peptide might not be required for partial dehydration by the modifying enzyme.

Dissecting reactions of nonlinear precursor peptide processing of the class III lanthipeptide curvopeptin.

Lanthipeptides are ribosomally synthesized peptides which undergo extensive post-translational modifications. In addition to novel structural features and bioactivities, the in vitro study on the biosynthesis of the class III lanthipeptide labyrinthopeptin revealed a unique C- to N-terminal directionality of biosynthetic processing. The recently described class III lanthipeptide curvopeptin allowed investigating the directionality aspect in much greater detail: Structural characterization of nine curvopeptin biosynthesis intermediates by high-resolution mass spectrometry combined with a deuterium-labeling approach enabled for the first time building a comprehensive biosynthesis model featuring all three post-translational modification reactions: phosphorylation, elimination, and cyclization. These results point to a nonlinear processing scheme with a predominant C → N-terminal directionality. Our data give important mechanistic insights into the concerted processing and directionality of the multifunctional class III modifying enzymes. The data are of significance in the light of obtaining a mechanistic understanding of the post-translational biosynthesis machinery of the growing variety of ribosomally synthesized and post-translationally modified peptides.