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

Abstract

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.

Mechanistic Understanding of Lanthipeptide Biosynthetic Enzymes

“…130 The in vitro reconstitution of LabKC demonstrated another unusual feature, the requirement of guanosine triphosphate (GTP) or deoxy-GTP for dehydration instead of ATP that is utilized by class II LanM enzymes. 477 Subsequent studies on a series of LanKC enzymes showed that the nucleotide specificity is not uniform: EryKC involved in erythreapeptin biosynthesis is ATP dependent, 132 AciKC that generates catenulipeptin accepts ATP, GTP, CTP, or TTP as a cosubstrate, 130 and StaKC 484 and CurKC 131 responsible for stackepeptin and curvopeptin biosynthesis, respectively, can utilize all NTPs/dNTPs but prefer purine nucleoside triphosphates as the phosphoryl donor for the central kinase domain (for structures of these class III lanthipeptides, see Figure 48). …”

“…Phylogenetic analysis of class III enzymes that make Lan or Lab did not provide any clear indications that could be predictive of the outcome. 36 Even more interestingly, some LanKC enzymes such as EryKC 132 and StaKC 484 can form both Lan and Lab in the same product (e.g., Figure 48 for the product of EryKC). Although the molecular mechanism of how the enzymes achieve the remarkable tandem cross-linking reaction resulting in labionin is still mostly unclear, several insightful observations have been reported.…”

“…484 Thus, it may be that the leader peptide also functions as an allosteric activator as in many other RiPP biosynthetic enzymes. 150 Like the substrates for class I and II lanthipeptides, 257 and those for cyanobactins 491 and microcin B17 492 (two other RiPP families), the LabA2 leader peptide has a propensity to form an α-helical secondary structure.…”

Ribosomally synthesized and post-translationally modified peptide natural product discovery in the genomic era

“…Recent genome mining for lanthipeptides has been fruitful, leading to the discovery of bicereucin [27], the cerecidins [28], and stackepeptin [29], among others. An interesting BGC was discovered in Ruminococcus flavefaciens FD-1, a commensal bacterium occurring in the rumen of animals, that encoded two putative class II lanthipeptide synthetases and twelve putative lanthipeptide precursor peptides.…”

Eine amphipathische alpha‐Helix lenkt die Modifizierung ribosomal‐synthetisierter Lipolanthine

“…Obwohl frühere Studien nahelegen, dass LP von Klasse‐I Lanthipeptiden eine helikale Struktur annehmen, zeigen neueste Co‐Kristallisationen der Dehydratase NisB (Nisin) mit dem Vorläuferpeptid NisA, dass das LP vielmehr eine β‐Strang‐Konformation einnimmt, mit der es an einem dreisträngigen antiparallelen β‐Faltblatt in NisB bindet . Für Klasse‐III Lanthipeptide wird ein konserviertes, N‐terminales LLDLQ‐Motiv im LP von Stackepeptin und verwandten Lanthipeptiden vorgeschlagen . Müller et al.…”