Lantibiotics are ribosomally synthesized peptide antibiotics containing the amino acids lanthionine (Lan) and methyllanthione (MeLan) as the most important and characteristic post-translational modification.[1] Work on the in vitro characterization of the enzymatic processing of lantibiotics, in particular for the formation of lanthionine, was described in reports on lacticin 481 [2] and haloduracin.[3] Recently, we identified a new family of lantibiotics, named labyrinthopeptins, which are produced by the actinomycete Actinomadura namibiensis DSM 6313. [4][5][6] The structure elucidation of labyrinthopeptin A2 (Scheme 1) mainly performed by X-ray crystallography highlighted a new amino acid, named labionin (Lab) (Scheme 1). Labionin is a triamino acid with a 2S,4S,8R configuration, and consists of a central quarternary carbon atom with a lanthionine motif and an unusual methylene bridge, which establishes a covalent link to a further amino acid moiety. This structure facilitates the formation of two rings in a linear peptide. Bioactivity assays revealed that labyrinthopeptin A2 has an excellent efficacy against neuropathic pain in an in vivo mouse model (ED 50 = 50 mg kg À1 ). The additionally found labyrinthopeptins A1 and A3 have been proposed as analogues of the A2 structure containing different amino acid constituents and an alternated ring size of the lanthionine motif.[4]The overall identification and sequencing of the gene cluster revealed only five genes which could be assigned to the biosynthesis of labyrinthopeptins. These contained two structural genes (labA1/A2) as precursors of labyrinthopeptin A1/ A3 and A2, two genes with sequence similarity to ATPdependent ABC transporters for the putative peptide export (labT1 and labT2), and one gene (labKC) for a putative modifying enzyme.[4] LabKC (MW = 95 kDa) is a two-domain protein consisting of an N-terminally conserved domain with features of an eukaryotic Ser/Thr protein kinase and a C-terminal domain with low sequence homology to LanC cyclases. In comparison to LanC cyclases, important active site residues, such as the zinc binding motif identified for the nisin cyclase, are missing in LabKC. [7] Moreover, the amino acid sequence of LabKC shows high homology to the SapB modifying enzyme RamC and to sequences from gene clusters of a considerable number of sequenced actinomycete strains (Supporting Information). [4,8] Therefore, we assign labyrinthopeptins as type-III lantibiotics.Herein we present the first in vitro reconstitution of the pre-labyrinthopeptin A2 biosynthesis in which a C À C bond formation is catalyzed by LabKC. This reaction involves the unprecedented requirement of guanosine triphosphate (GTP) for the phosphorylation and dehydratation reaction of serines. In other in vitro syntheses of lantibiotics adenosine triphosphate (ATP) and not GTP is used. [2,3] From the obtained data a biosynthetic model has been deduced. To our knowledge, this is the first time that GTP has been shown to act as a cosubstrate in the formation of lanthionine-ty...
Lantibiotics are peptide antibiotics, realizing their unique secondary structure by posttranslational modifications, the most important one being the formation of the characteristic amino acid lanthionine. Like other ribosomal peptide antibiotics, they are synthesized with an N-terminal leader peptide important for posttranslational processing by modifying enzymes; after peptide maturation, the leader peptide is proteolytically cleaved off. Numerous studies of the leader peptides of class I and II lantibiotics already showed their crucial role in recognition, self-immunity, and extracellular transport. The recently described labyrinthopeptins, members of the family of class III lantibiotics, exhibit the characteristic novel amino acid labionin, which was revealed by elucidation of the structure of labyrinthopeptin A2. The assembly of the labionin motif in the linear peptide chain is mediated by the lyase-kinase-cyclase-type enzyme LabKC through a serine side chain phosphorylation with GTP, elimination of the phosphate group, and a subsequent 2-fold Michael-type addition cyclization. In this work, we systematically investigated for the first time the importance of the leader peptide in the processing of class III lantibiotics using the example of the labyrinthopeptin A2 precursor peptide. In vitro studies with synthetic leader peptide analogues revealed that a conserved N-terminal hydrophobic patch on a putative helical structure is required for the proper peptide processing by the modifying enzyme LabKC. On the other hand, studies showed that the C-terminal part of the leader peptide serves as a spacer between the binding site and active sites for phosphorylation and elimination, thus restricting the number of hydroxy amino acid side chains that could undergo dehydration. Finally, a model for the peptide recognition and processing by the LabKC has been postulated.
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.
The biosynthesis of a considerable number of ribosomally synthesized peptide antibiotics involves the modification of Ser and Thr residues of a precursor peptide. This post-translational processing is performed by one or multiple modifying enzymes encoded in the biosynthetic gene cluster. We present a deuterium-label based enzyme assay, utilizing a series of peptide substrates with α-deuterated Ser, for the determination of the dehydration order during the biosynthesis of class III lantibiotic labyrinthopeptin A2. Remarkably, the data show that, in contrast to other modifying enzymes of class I and II lantibiotics, LabKC has a C- to N-terminal processing mode. This surprising finding, which we consider relevant for the biosyntheses of other class III lantibiotics, underlines significant differences of this class of modifying enzymes compared to other investigated systems.
Lanthipeptides represent an important group of ribosomally synthesized and post-translationally modified peptides (RiPPs). Commonly, in the last steps of their maturation, a part of the peptide, termed the leader, is removed, providing the active compound. This contribution describes for the first time the identification of a protease involved in the removal of the leader peptide of a class III lanthipeptide. Four putative class III biosynthetic gene clusters were identified in bacterial genomes, each containing a gene encoding a prolyl oligopeptidase (POP). Further in vitro investigations of the gene cluster from Kribbella flavida , involving reconstitution of the biosynthesis of the new lanthipeptide flavipeptin, proved that a POP-type FlaP protease is responsible for leader removal. Interestingly, detailed in vitro studies of the substrate specificity revealed that FlaP is specific to the post-translationally modified peptide and can discriminate between N- and C-terminal rings. Therefore, it has been shown for the first time that factors other than size and amino acid sequence might be involved in substrate recognition by POPs.
Lantibiotics are ribosomally synthesized peptides containing post-translationally installed lanthionine thioether bridges. Recently characterized class III lantibiotics have also revealed the occurrence of labionin, a novel carbacyclic variation of lanthionine, and highlighted the structural diversity within this group. Here we describe the discovery and characterization of curvopeptins produced by Thermomonospora curvata, the first class III lantibiotics of thermophilic origin. Furthermore, investigation of the modifying enzyme CurKC and in particular the characterization of its specificity toward phosphorylation co-substrates was performed. Remarkably, all investigated NTPs and dNTPs were accepted by the enzyme, although the purine nucleotides ATP/dATP and GTP/dGTP were the preferred co-substrates. This finding complements previous studies on the class III lantibiotic synthetases LabKC and EryKC and underlines the surprising promiscuity of the Ser/Thr-kinase domain. Enzymatic studies with a precursor peptide mutant allowed the assignment of all dehydration sites and further GC-MS analysis revealed the presence of lanthionine as the main type of intramolecular ring.
American foulbrood (AFB) caused by the bee pathogenic bacterium Paenibacillus larvae is the most devastating bacterial disease of honey bees worldwide. From AFB-dead larvae, pure cultures of P. larvae can normally be cultivated indicating that P. larvae is able to defend its niche against all other bacteria present. Recently, comparative genome analysis within the species P. larvae suggested the presence of gene clusters coding for multi-enzyme complexes, such as non-ribosomal peptide synthetases (NRPSs). The products of these enzyme complexes are known to have a wide range of biological activities including antibacterial activities. We here present our results on antibacterial activity exhibited by vegetative P. larvae and the identification and analysis of a novel antibacterially active P. larvae tripeptide (called sevadicin; Sev) produced by a NRPS encoded by a gene cluster found in the genome of P. larvae. Identification of Sev was ultimately achieved by comparing the secretome of wild-type P. larvae with knockout mutants of P. larvae lacking production of Sev. Subsequent mass spectrometric studies, enantiomer analytics and chemical synthesis revealed the sequence and configuration of the tripeptide, D-Phe-D-ALa-Trp, which was shown to have antibacterial activity. The relevance of our findings is discussed in respect to host-pathogen interactions.
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