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.

Elucidation of sevadicin, a novel non-ribosomal peptide secondary metabolite produced by the honey bee pathogenic bacterium Paenibacillus larvae.

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.

Elucidation of sevadicin, a novel non-ribosomal peptide secondary metabolite produced by the honey bee pathogenic bacterium Paenibacillus larvae.

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.

Champacyclin, a new cyclic octapeptide from Streptomyces strain C42 isolated from the Baltic Sea.

New isolates of Streptomyces champavatii were isolated from marine sediments of the Gotland Deep (Baltic Sea), from the Urania Basin (Eastern Mediterranean), and from the Kiel Bight (Baltic Sea). The isolates produced several oligopeptidic secondary metabolites, including the new octapeptide champacyclin (1a) present in all three strains. Herein, we report on the isolation, structure elucidation and determination of the absolute stereochemistry of this isoleucine/leucine (Ile/Leu = Xle) rich cyclic octapeptide champacyclin (1a). As 2D nuclear magnetic resonance (NMR) spectroscopy could not fully resolve the structure of (1a), additional information on sequence and configuration of stereocenters were obtained by a combination of multi stage mass spectrometry (MSn) studies, amino acid analysis, partial hydrolysis and subsequent enantiomer analytics with gas chromatography positive chmical ionization/electron impact mass spectrometry (GC-PCI/EI-MS) supported by comparison to reference dipeptides. Proof of the head-to-tail cyclization of (1a) was accomplished by solid phase peptide synthesis (SPPS) compared to an alternatively side chain cyclized derivative (2). Champacyclin (1a) is likely synthesized by a non-ribosomal peptide synthetase (NRPS), because of its high content of (D)-amino acids. The compound (1a) showed antimicrobial activity against the phytopathogen Erwinia amylovora causing the fire blight disease of certain plants.

Involvement and unusual substrate specificity of a prolyl oligopeptidase in class III lanthipeptide maturation.

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.

Curvopeptin: a new lanthionine-containing class III lantibiotic and its co-substrate promiscuous synthetase.

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.

Deuterium labeled peptides give insights into the directionality of class III lantibiotic synthetase LabKC.

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.

Leader peptide-directed processing of labyrinthopeptin A2 precursor peptide by the modifying enzyme LabKC.

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.