The Supersized Class III Lanthipeptide Stackepeptin Displays Motif

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The supersized class III lanthipeptide stackepeptin displays motif multiplication in the core peptide Natalia A. Jungmann, Eric F. van Herwerden, Manuela Hügelland, and Roderich D. Süssmuth ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00651 • Publication Date (Web): 21 Oct 2015 Downloaded from http://pubs.acs.org on October 24, 2015

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ACS Chemical Biology

The supersized class III lanthipeptide stackepeptin displays motif multiplication in the core peptide Natalia A. Jungmann,a Eric F. van Herwerden,a Manuela Hügellanda and Roderich D. Süssmuth*a a

Fakultät II-Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 124, 10623 Berlin, Germany

KEYWORDS. Lanthipeptides, stackepeptin, directionality, post-translational modification, lanthionine, labionin

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 DSM44728T. The stackepeptins are the first supersized class III lanthipeptides to be discovered. Unlike other class III lanthipeptides they consist of three lanthionine/labionine moieties instead of two. In this study both, in vivo and in vitro maturation of the peptides has 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 towards the phosphorylating co-substrate 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.

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Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly expanding group of structurally diverse natural products.1 The remarkable structural heterogeneity among these peptides is due to post-translational modifications (PTMs) introduced by specific enzymes. One of the most studied classes of RiPPs synthesized by bacteria is the group of lanthipeptides - polycyclic peptides bearing the characteristic cross-linked diamino diacids lanthionine (Lan) and methyllanthionine (MeLan). These intramolecular thioether bridges provide protection against proteolytic degradation and give lantipeptides a wide range of bioactivities and cellular roles.2,3 Lanthipeptides are divided into four classes according to which types of enzymes are responsible for their posttranslational processing. With classes I to IV, the processing enzymes can install Lan/MeLan moieties. These are formed by dehydration of Ser/Thr residues to 2,3-didehydroalanine (Dha) and 2,3-didehydrobutyrine (Dhb), followed by a Michael-type addition of a Cys residue to Dha/Dhb. With class III, the processing enzymes can also install (methyl)labionin (Lab/MeLab), a triamino triacid consisting of both a thioether- and carbon bridge. Here, two Ser/Thr residues are dehydrated and together with a Cys residue undergo a double Michael addition through an as of yet unknown mechanism.4,5 The application of genome mining approaches allows for the identification and characterization of novel natural products even before their isolation.6,7,8 For RiPPs, genome mining and biosynthetic predictions are simplified by the fact that the gene clusters of a specific subgroup have very similar architecture. In this context the aerobic, non-pathogenic, Gram-positive Actinomycete Stackebrandtia nassauensis DSM44728T attracted our attention. It was isolated from a soil sample collected on the roadside in Nassau, Bahamas.9 To date, only one additional species of the genus Stackebrandtia is known, namely S. albiflava, which has been isolated from a soil sample in the tropical rainforest in China. A preliminary analysis of the genome sequence revealed genes associated with secondary metabolite biosynthesis, transport and catabolism. This makes S. nassauensis a potential source of novel natural products.10 In this work we present stackepeptin A and its variants as novel class III lanthipeptides from Stackebrandtia nassauensis DSM-44728T. Natural products observed from in vivo experiments were mass spectrometrically characterized. Furthermore, in vitro biosynthesis of the stackepeptins was explored via heterologous expression of the modifying enzyme StaKC.

RESULTS AND DISCUSSION Class III lanthipeptides from Stackebrandtia nassauensis DSM-44728T. Genome mining of S. nassauensis revealed a putative class III lanthipeptide gene cluster. According to a BLAST search, the 2 ACS Paragon Plus Environment

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modifying enzyme StaKC (GenBank accesion number ADD40246.1) has query coverage of 98–99% and identity of 45–50% to other class III lanthipeptide modifying enzymes LabKC and CurKC, respectively. StaKC has a protein architecture which is characteristic for this class of enzymes: it consists of a putative N-terminal lyase, a central Ser/Thr kinase and a C-terminal cyclase domain.12 As expected, we could not identify zinc-binding amino acids in the cyclase domain. The N-terminal leader sequence of the precursor peptide contains the conserved hydrophobic patch motif “LLDLQ”, which is a known binding motif for class III lanthipeptides.12 A manual ORF search in the downstream region revealed two genes encoding putative ABC-transporters and one gene encoding a LuxR-family transcriptional regulator (Figure 1B). All abovementioned features are clear indications for a class III lanthipeptide gene cluster (sta cluster). The putative gene staA encodes a 50-amino acid precursor peptide. Remarkably, this putative core peptide unexpectedly contained three Ser-(Xxx)2-Ser-(Xxx)2-5-Cys motifs (Xxx = random amino acid) as opposed to the common two motifs seen in all other known class III lanthipeptides (Figure 1).13,14,15,16 This implicated the presence of three lanthionine or labionine moieties. Because the putative core region contains two additional serines and two threonines, the mature peptide could theoretically display up to 10 dehydrated residues. In order to differentiate between Lan formation resulting in one macrolactam ring (e.g. ring A) and Lab formation resulting in two macrolactam rings (e.g. rings A/A’), we suggest to employ the term “segment” for the posttranslationally cyclized Ser-(Xxx)2-Ser-(Xxx)2-5-Cys motif. To evaluate the production of the bioinformatically predicted lanthipeptide, we investigated extracts of cultures grown on various media by mass spectrometric screening (Figure S4). Our previous studies on class III lanthipeptides taught us also to consider the absence of specific cleavage sites for leader peptide removal.15 Therefore, we searched HPLC-MS chromatograms for various predicted peptide masses with different dehydration states and with different N-terminal proteolytic processing. While liquid culture media did not render any hits, we detected two triply charged ions of [M+3H]3+ = 901.4087 and [M+3H]3+ = 882.4023 in the mass spectra of cultures grown on solid Gym-Strep-medium. The first ion corresponds to 8-fold dehydrated stackepeptin A after removal of the 19 N-terminal amino acids ([Mcalc.]3+ = 901.4061, mass error = 2.9 ppm). The second ion corresponds to the same peptide lacking one Nterminal Gly residue ([Mcalc.]3+ = 882.3990, mass error = 3.7 ppm; Figure 2). MS/MS fragmentation of these peptides was consistent with the formation of three (Lan-type) to six (Lab-type) rings (Figure 2 and S3). Moreover, the analysis showed that all serines in the A-segment are dehydrated, whereas in the Band C-segments either one Ser or one Thr must have escaped modification. Furthermore, two more stackepeptins were detected in cultures that were grown on different media (Figure S4 and Table S5). These peptides, designated as stackepeptin C and D, contained either seven or six dehydrations, 3 ACS Paragon Plus Environment


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respectively. Subsequent MS/MS experiments showed that in the case of stackepeptin C, one residue in the C-terminal segment escaped dehydration and for stackepeptin D, one residue in both the B- and Csegments had not been modified (Figure 2 and Figure S3). It is important to note that for two particular media the only difference in the stackepeptin A producing medium (Gym-Strep) and stackepeptin C and D producing medium (HA) was the presence of CaCO3. We performed several experiments that aimed to identify the exact role of calcium carbonate, but for none of the tested conditions production of stackepeptin C and D was decreased (Figure S5). Therefore, we were unable to find an explanation for the production of stackepeptin C and D and further studies would be required.

Structure of stackepeptins. The above described MS/MS analysis proved that all stackepeptins possess three segments with non-overlapping rings that contain the Ser/Ser/Cys precursor motifs (Ser3 to Cys10, Ser12 to Cys19 and Ser23 to Cys30). We then determined which Ser/Thr residues are dehydrated and whether either a Lab or a Lan + Dha moiety is present. While we previously reported on a GC-MS method to identify labionin,18 this approach gives no information on the positions of Lan/Lab within the peptide (segment A, B or C). Therefore, we instead carried out chemical derivatization of the natural products with β-mercaptoethanol (βME) to identify free Dha's.14,19 MS/MS analysis of the treated peptides then allowed us to identify the non-fragmented peptide rings and localize where βME additions had taken place, hence distinguish between Lab and Lan + Dha. Upon incubation of stackepeptins A, C and D with βME, addition of one and two βME moieties was observed. Subsequent MS/MS analysis showed that these adducts were exclusively located in the Asegment (Figure S6), meaning that for all three peptides, segment A contains free Dha. Accordingly, stackepeptin A (1) must contain three labionins, stackepeptin C (3) two labionins (segments A and B) and one lanthionine (ring C), and stackepeptin D (4) one labionin (segment A) and two lanthionines (rings B and C). The presence of the unmodified serines in the center of the Lan ring is a novel feature of stackepeptins C and D among class III lanthipeptides; with all so far known class III lanthipeptides bearing Lan, e.g. SapB17, erythreapeptin15 and curvopeptin,16 these serine residues are always dehydrated. Our previous work on the in vitro reconstitution of reactions of LanKC with Ser→Ala variant peptides of curvopeptin showed that conversion of the central Ser to Dha is essential for further processing and Lan formation in curvopeptin.20 Apparently StaKC has a higher sequence tolerance than other LanKC enzymes characterized so far. This might also imply that every LanKC has its own structural requirements for substrate processing.


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The last question that remained was whether the Ser or Thr residues in segments B and C (Ser15 or Thr17 and Thr22 or Ser23, respectively) were processed. While we were not able to answer this question for segment B at this point in time, the MS/MS data (Figure S6) suggest that Thr22 in segment C escaped dehydration. Further evidence for this was found during the in vitro experiments.

In vitro reconstitution of StaA processing by StaKC. An important approach to characterization of the biosynthetic activity of enzymes involved in RiPP pathways is the heterologous expression of modifying enzymes and subsequent in vitro reaction with the precursor peptide.21–23 For this purpose, the modifying enzyme construct His6-StaKC was heterologously co-expressed with genes encoding chaperones GroES and GroEL on the commercially available plasmid pGro7 in E. coli BL21Gold(DE3) (see Supporting Information). The chaperones proved to be necessary due to solubility or activity issues we encountered when we attempted to express only the His6-StaKC or as various fusion protein constructs. Correct folding of the modifying enzyme was confirmed by CD spectroscopy (Figure S2). After purification, the enzyme was incubated with synthetic precursor peptide StaA in the presence of Mg2+ and ATP (for purification protocols see Supporting Information). Already after 1 h full conversion to the 8-fold dehydrated peptide with leader attached, termed modified StaA, was observed (Figure 3). This confirmed the dehydratase activity of StaKC. For further characterization, modified StaA was subjected to ESI-MS3 analysis. A fragmentation pattern of the core peptide was observed which was similar to that of stackepeptin A (Figure S7). This suggested that three cyclized segments had been formed. To assess whether all rings were installed in the same manner as in the natural product, chemical derivatization of the processed precursor peptide was carried out. Treatment of the modified StaA with iodoacetamide (IAA) showed no reaction with free cysteines, which confirmed that all rings had been formed (Figure S8A). In a second control reaction the peptide was derivatized with βME. Three βME additions were observed, indicating the presence of three Dha residues that were not involved in ring formation (Figure S8A). Subsequent MS/MS analysis revealed that all three additions were located in the A-segment. This shows that in vitro generated modified StaA consists of an N-terminal Lan ring with three Dha and two C-terminal Lab rings, as opposed to natural stackepeptin A, where three Lab rings and two Dha were found. Owing to the discrepancy between in vivo and in vitro topology of the N-terminal ring, the question needed to be addressed whether a Lab could be installed in the region of segment A by in vitro experiments. Variation of reaction parameters (increased enzyme or ATP concentration, variation of pH) was explored, but any experiment with modified reaction conditions still rendered Lan in segment A 5 ACS Paragon Plus Environment


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(data not shown). We then argued that the abundance of glycines in the region of segment A might increase flexibility of the peptide backbone. This in turn might impede the enzyme to bring the precursor peptide into the optimal conformation required for Lab formation. To test this idea, a synthetic peptide in which the amino acid sequence of segment A had been exchanged with that of segment B was incubated with StaKC, followed by chemical derivatization with IAA and βME (Figure S10). Mass spectrometric analysis of the reaction showed no adduct formation after either experiment. Furthermore, we were able to identify all three Lab fragments with MS/MS. This confirms that incomplete N-terminal in vitro processing is inherently encoded in the sequence of segment A. As to why this only causes difficulties for in vitro processing, one could argue that for example in vivo processing is facilitated by protein-protein interactions within the cell, e.g. with the transporter proteins StaT1/T2 as a part of the stackepeptin biosynthesis machinery.

Purine nucleotides are preferred phosphate donors for StaKC. Because a promiscuous use of phosphate donors is known to occur within LanKC enzymes14,15,16, we also tested if StaKC is able to use nucleotides other than ATP. StaKC did indeed show promiscuity towards potential co-substrates. However, unlike other class III synthetases, StaKC shows strong preference for purine nucleotides. In reactions where purine nucleotides were used, exclusively the eightfold dehydrated product was observed, whereas reactions with pyrimidine nucleotides led to a range of differently dehydrated products (Figure S11). Then we were interested to see if shortening the reaction times with purine (d)NTP would also yield the same intermediates which were observed for pyrimidine nucleotides. ATP was chosen as the co-substrate in this experiment. As shown in Figure S12, no significant accumulation of side products was observed, despite significantly shorter reaction times (2–15 min). This indicates that purine nucleotides facilitate rapid processing and formation of uniform products by StaKC. The presence of diverse composition of dehydrated products while employing pyrimidine nucleotides (Figure S11) indicates that processing is slowed down or even hampered.

Ser/Thr→Ala-StaA variants reveal the in vitro directionality of StaKC. Having established an in vitro system for StaA enabled further assessment of the importance of particular amino acid residues within the core peptide for StaKC processing. First, we tested the two variant peptides StaA-T17A and StaA-T22A to attain conclusive evidence that those two amino acids escape dehydration upon incubation with StaKC. As can be seen in Figure 4 and Figure S8B-C after 2 h of incubation, 8-fold dehydrated peptides were detected as main products, thus indicating that neither Thr residues are processed by StaKC. Detailed analysis of the MS/MS product ion spectra of the corresponding βME-derivatized 6 ACS Paragon Plus Environment

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peptides showed three additions in the A-segment as previously found for modified StaA (Figure S9A-C). To confirm these results and to gain more insight into the role of Ser and Cys residues in ring formation we prepared seven synthetic peptides with Ser→Ala and Cys→Ala exchanges: S3A/S6A, S4A/S9A, S12A, S15A, S23A, S26A, and C10A/C19A/C30A. The peptide lacking all Cys residues (StaA-C10A/C19A/C30A) was a very poor substrate to His6-StaKC; after 18 h only intermediates bearing two phosphorylations and three to five Dha residues were observed (Figure S13).This was an unexpected result, as the double Cys→Ala variant peptides of curvopeptin16 and flavipeptin24 were fully dehydrated. To see if the C-terminus is as important for stackepeptin processing as for other class III lanthipeptides,14,20 we examined the impact of Ser→Ala mutations in this region. Reactions with StaKC of both variant peptides, StaA-S23A and StaA-S26A, showed impaired processing as only intermediates bearing multiple phosphorylations with additional dehydrations were observed (Figure S8H–I). This strongly suggests that formation of the C-terminal ring occurs at the beginning of the in vitro stackepeptin maturation and is indispensable for further processing of single residues. We then examined the importance of Ser in the A- and B-segments. All StaA variant peptides (S3A/S6A, S4A/S9A, S12A and S15A) were found to be fully dehydrated by StaKC. As can be seen in Figure 4 and Figure S8D–G the variant peptides S12A and S15A were 7-fold dehydrated, whereas the double variants S3A/S6A and S4A/S9A were only 6-fold dehydrated. This further confirms that Thr residues are not being modified by StaKC. We additionally investigated ring formation by IAA and positioning of Dha residues by βME derivatization. From studies on catenulipeptin14 and curvopeptin20 it was already known that exchange of any of the Ser residues in the N-terminal part abolishes formation of the N-terminal ring. However, as can be seen in Figures 4 and S8 for all tested fully dehydrated variant peptides the major product lacks IAA adducts. The results of derivatization with IAA, apart from some minor by-products (see Figure S8BG), are fully consistent with the number of observed βME adducts. For Thr→Ala (StaA-T17A and StaAT22A) variants as well as for Ser→Ala variants in the B-segment (StaA-S12A and StaA-S15A) three βME adducts were observed, whereas for Ser→Ala variants in the A-segment (StaA-S3A/S6A and StaAS4A/S9A) only one βME adduct as a major product was observed. Presence of the y21-ion in the MS/MS spectra of βME-derivatized peptides revealed that all additions had occurred within the A-segments. Interestingly, mass spectra of the StaA-S12A peptide indicate lanthionine formation of unusual size (segment B). For the non-mutated modified StaA a labionin is installed in this segment, suggesting that formation of this small lanthionine ring in the StaA-S12A might reflect an intermediate from hindered labionin formation. The ability of StaKC to form rings (A- and B-segments) in the N-terminal part in spite of Ser→Ala exchanges is in contrast to previously described class III lanthipeptides where the processing 7 ACS Paragon Plus Environment


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of the N-terminus was hampered.14,20 This feature of StaKC is of interest for peptide engineering studies as more thorough modifications of the peptide backbone could be performed. Furthermore, studies on Ala-variant peptides showed a pronounced dependence on proper C-terminal labionin formation in in vitro StaKC approaches, which is in contrast to a variable Lan or Lab formation during in vivo maturation (Figure 5). While variant peptide S15A is fully processed, which appears to be in line with non-processed Ser in stackepeptin D in this position, processing of peptide S26A is hampered (Figures 4 and 5) which underlines necessity for an interaction with the Ser side chain.

The core peptide is partially processed in the absence of the leader. Previous studies on class I and II lanthipeptides showed that the presence of the leader peptide is not strictly required for an initial modification of the core peptide.25,26 More recent studies on haloduracin show that there is a synergism in binding of core and leader peptide, however the leader binds with higher affinity than the core and increases the affinity of the latter to the modifying enzyme.27 In the case of class III lanthipeptides, in trans addition of the leader peptide either rendered no (LabA212) or very poor (catenulipeptin14) conversion of the core peptide and no conversion was observed when only the core peptide was tested. We first tested the ability of StaKC to process the core peptide only, and observed a singly dehydrated product (Figure S14B). This is the first example of a class III lanthipeptide synthetase that processes the core peptide in absence of the leader. We then examined if in trans addition of the leader peptide would lead to a higher degree of processing. This experiment yielded peptides with up to two dehydrations. The MS/MS analysis of the most abundant singly dehydrated peptide ion indicated dehydration of one of the C-terminal serines (Figure S14). This is in line with the results of Ser→Ala variants and shows that in vitro biosynthesis of stackepeptin starts C-terminally. The additional dehydration event found in the in trans experiment supports the hypothesis that the leader peptide indeed promotes the activity of the modifying enzyme.27 If one considers the hypothesis of various enzyme conformations as expressed for LctM,24 these experiments showed that StaKC might already be in a partially active state which is slightly enhanced by the binding of the leader peptide.

Conclusions. Employing a genome mining approach we discovered stackepeptins, new supersized members of the class III lanthipeptides produced by Stackebrandtia nassauensis DSM-44728T. These peptides possess one more Ser/Ser/Cys motif than all class III lanthipeptides known to date. By variation of the culture medium we could isolate and characterize three stackepeptins with various ring topologies (Lab+2Dha/Lab/Lab, Lab+2Dha/Lab/Lan and Lab+2Dha/Lan/Lan). Furthermore, we were able to reconstitute and characterize the in vitro biosynthesis of stackepeptin. Interestingly, the ring topology of 8 ACS Paragon Plus Environment

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the final 8-fold dehydrated in vitro product (Lan+3Dha/Lab/Lab) differs from natural stackepeptin A (Lab+2Dha/Lab/Lab). Our experiments suggest that segment A is more difficult to process as replacing its sequence with that of segment B resulted in the formation of three labionins. This could imply that in vivo additional yet unknown factors assist in processing of StaA. Also of interest is that in in vitro reactions StaKC partially modifies the core peptide if the leader is missing and its activity slightly increases with addition of the leader in trans. Studies on Ser→Ala variant peptides of StaA showed a strong dependence on the C-terminal labionin formation for full processing, which together with the results of fragmentation of the in trans dehydrated core peptide indicates that in vitro processing starts at the C-terminus. The finding of a triple Ser/Ser/Cys motif in stackepeptins implies that class III lanthipeptides may have evolved from a common precursor peptide with only one segment-containing precursor peptide by duplication or multiplication of segments. This idea of sequence multiplication has been previously found in cyanobactins: for patellamides, trunkamides and tenuecyclamides double, triple and even quadruple motives have been identified.28 Perhaps sequence multiplication is more frequently occurring across the RiPPs than previously thought, and might be observed in future genome mining studies. In summary, herein described stackepeptins extend the structural diversity of the family of class III lanthipeptides by new and unusual representatives. This underlines the importance for a thorough characterization of in vivo and in vitro maturation in order to obtain a comprehensive picture of the lanthipeptide biosynthesis as an important group of RiPPs.

METHODS Isolation and purification of stackepeptins from S. nassauensis DSM-44728T. Components of growth media were purchased from Sigma Aldrich, Carl Roth GmbH, Becton Dickinson and Archer Daniels Midland Company. S. nassauensis was grown on DSMZ-agar medium No. 65 (here also named Gym-Strep-medium) and also on the medium No. 65 lacking CaCO3 (here also named HA-medium) at 28°C.

For

detailed

recipes:

https://www.dsmz.de/catalogues/catalogue-microorganisms/culture-

technology/list-of-media-for-microorganisms.html. After 3–10 days stackepeptins were extracted from plates with acetonitrile. Dried extract was resuspended in water and extracted with butanol. The obtained dried extract was resuspended in 15 % aqueous methanol solution (0.1 % formic acid) and loaded onto 40 g C18 wide pore flash cartridge provided by Grace Division. Flash chromatographic separation was carried out on a RevelerisX2 flash system (Grace Divison) using a mobile phase that 9 ACS Paragon Plus Environment


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consisted of H2O (A) and methanol (B) each containing 0.1 % formic acid with the following gradient 15– 60 % B over 15 min, 60–99 % over 5 min, than hold for 3 min, yielding stackepeptin-containing fractions that were concentrated using centrifugal evaporation followed by freeze-drying of the aqueous phase. The prepared samples were used for βME derivatization studies (ca. 0.2 mg peptide was used). For further purification of stackepeptin A produced on DSMZ-agar No. 65 separation on an analytical HPLC system (Agilent Technologies 1100 Series) was carried out. A gradient 20–40 % B (where B is acetonitrile and A is H2O with 0.1 % formic acid each) over 10 minutes on the Luna 5u C18(2) 100A (100x4.60 mm, Phenomenex) column was used. The purity of the extracted stackepeptin is shown in Figure S15.

Cloning and Protein Expression. Detailed procedures are described in the Supporting Information. Solid-Phase Peptide Synthesis of StaA precursors. Detailed procedures are described in the Supporting Information.

Enzyme activity assay. Enzymatic assays and peptide synthesis was performed according to protocols reported previously by Müller et al.11 The reaction mixture consisted of 20 mM TES buffer (pH 7.0), 10 mM MgCl2, 1 mM dithiothreitol, 1 mM NTP, 5 µM His6-StaKC and 75 µM precursor peptide. After incubation at 28°C for 1 h (or longer if stated) the reaction was quenched with one volume equivalent acetonitrile (1:1, v:v). If required, subsequent incubation with βME (6 mM, 2 h at 28 °C) or IAA (30 mM in Tris/HCl buffer (pH 8.3–8.5), in the dark) was conducted.

HPLC-ESI-MS and HPLC-ESI-MS/MS experiments. All HPLC-ESI-MS and HPLC-ESI-MS/MS analyses were conducted using an LTQ-Orbitrap XL (Thermo Fisher Scientific) coupled to a HPLC system 1200 (Agilent Technologies). Chromatographic separations were performed using GromSil 120 ODS-5 ST columns (50×2.0 mm, Grace) with a linear mobile phase gradient consisting of solvent A (0.1 % formic acid in water) and solvent B (0.1 % formic acid in acetonitrile). The gradient was 5−100% B over 10 min. ESI-MS/MS experiments were recorded in FTMS mode with a resolution R = 7,500. For fragmentation of the peptide ions by means of MS/MS or MS3 methods collisionally-induced dissociation was performed (normalized collision energy of 40%). Data were analyzed using the Thermo Xcalibur 2.2 software.

Associated content Supporting Information. Detailed experimental procedures and mass spectrometric data. This material is available free of charge via the Internet at http://pubs.acs.org.


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Author Information Corresponding Author * [email protected]

Author Contributions All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

Funding Sources This research was supported by grants from the Deutsche Forschungsgemeinschaft (DFG SU239/8-1), the Cluster of Excellence “Unifying Concepts in Catalysis” coordinated by the Technical University Berlin and the EU project StrepSynt (FP7-KBBE 613877). N.A.J. acknowledges a fellowship from the Fonds der Chemischen Industrie.

Acknowledgements We thank A. Mainz for helpful discussions and critical review of the manuscript.

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13. Meindl, K., Schmiederer, T., Schneider, K., Reicke, A., Butz, D., Keller, S., Gühring, H., Vértesy, L., Wink, J., Hoffmann, H., Brönstrup, M., Sheldrick, G. M., and Süssmuth, R. D. (2010) Labyrinthopeptins: A new class of carbacyclic lantibiotics, Angew. Chem. Int. Ed. 49, 1151–1154. 14. Wang, H., and van der Donk, W. A. (2012) Biosynthesis of the class III lantipeptide catenulipeptin, ACS Chem. Biol. 7, 1529–1535. 15. Völler, G. H., Krawczyk, J. M., Pesic, A., Krawczyk, B., Nachtigall, J., and Süssmuth, R. D. (2012) Characterization of new class III lantibiotics - erythreapeptin, avermipeptin and griseopeptin from Saccharopolyspora erythraea, Streptomyces avermitilis and Streptomyces griseus demonstrates stepwise N-terminal leader processing, ChemBioChem 13, 1174–1183. 16. Krawczyk, B., Völler, G. H., Völler, J., Ensle, P., and Süssmuth, R. D. (2012) Curvopeptin: A new lanthionine-containing class III lantibiotic and its co-substrate promiscuous synthetase, ChemBioChem 13, 2065–2071. 17. Kodani, S., Hudson, M. E., Durrant, M. C., Buttner, M. J., Nodwell, J. R., and Willey, J. M. (2004) The SapB morphogen is a lantibiotic-like peptide derived from the product of the developmental gene ramS in Streptomyces coelicolor, Proc. Natl. Acad. Sci. U. S. A. 101, 11448–11453. 18. Pesic, A., Henkel, M., and Süssmuth, R. D. (2011) Identification of the amino acid labionin and its desulfurised derivative in the type-III lantibiotic LabA2 by means of GC/MS, Chem. Commun. 47, 7401–7403. 19. Walk, T. B., Süssmuth, R. D., Kempter, C., Gnau, V., Jack, R. W., and Jung, G. (1999) Identification of unusual amino acids in peptides using automated sequential Edman degradation coupled to direct detection by electrospray-ionization mass spectrometry, Biopolymers 49, 329–340. 20. Jungmann, N. A., Krawczyk, B., Tietzmann, M., Ensle, P., and Süssmuth, R. D. (2014) Dissecting reactions of nonlinear precursor peptide processing of the class III lanthipeptide curvopeptin, J. Am. Chem. Soc. 136, 15222–15228. 21. Xie, L., Miller, L. M., Chatterjee, C., Averin, O., Kelleher, N. L., and van der Donk, W. A. (2004) Lacticin 481: In vitro reconstitution of lantibiotic synthetase activity, Science 303, 679–681. 22. Li, Y.-M., Milne, J. C., Madison, L. L., Kolter, R., and Walsh, C. T. (1996) From peptide precursors to oxazole and thiazole-containing peptide antibiotics: Microcin B17 synthase, Science 274, 1188–1193. 23. Piwowarska, N. A., Banala, S., Overkleeft, H. S., and Süssmuth, R. D. (2013) Arg-Thz is a minimal substrate for the Nα,Nα-arginyl methyltransferase involved in the biosynthesis of plantazolicin, Chem. Commun. 49, 10703–10705. 13 ACS Paragon Plus Environment


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24. Völler, G. H., Krawczyk, B., Ensle, P., and Süssmuth, R. D. (2013) Involvement and unusual substrate specificity of a prolyl oligopeptidase in class III lanthipeptide maturation, J. Am. Chem. Soc. 135, 7426–7429. 25. Levengood, M. R., Patton, G. C., and van der Donk, W. A. (2007) The leader peptide is not required for post-translational modification by Lacticin 481 synthetase, J. Am. Chem. Soc. 129, 10314–10315. 26. Khusainov, R., and Kuipers, O. P. (2012) When the leader gets loose: In vivo biosynthesis of a leaderless prenisin is stimulated by a trans-acting leader peptide, ChemBioChem 5, 2433–2438. 27. Thibodeaux, G. N., McClerren, A. L., Ma, Y., Gancayco, M. R., and van der Donk, W. A. (2015) Synergistic binding of the leader and core peptides by the lantibiotic synthetase HalM2, ACS Chem. Biol. 10, 970–977. 28. Donia, M. S., Ravel, J., and Schmidt, E. W. (2008) A global assembly line to cyanobactins, Nat. Chem. Biol. 4, 341–343.


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Figure Legends Figure 1. A. Structural diversity of class III lanthipeptides. B. Biosynthetic gene cluster of stackepeptin with the sequence of the precursor peptide (recognition motif required for modification underlined, residues undergoing posttranslational modifications highlighted in orange). C. Alignment of type III lanthipeptide precursor peptides. Conserved amino acids are highlighted. Figure 2. A. Proposed structures of stackepeptin A (1) and stackepeptin B (2). B. Structures of lanthipeptide characteristic amino acids labionin and lanthionine. C. LC-MS spectra of triply charged stackepeptins produced by S. nassauensis grown on two different media (upper panel – DSMZ medium No. 65, lower panel - same medium lacking CaCO3). D. Fragmentation patterns of 8-, 7- and 6-fold dehydrated natural stackepeptin A (1), C (3), and D (4) (y-ions are highlighted in blue color, b-ions are marked in red color; fragmentation spectra are presented in SI, Figure S3). Figure 3. Scheme of the in vitro reaction of synthetic precursor peptide StaA with heterologously expressed His6-StaKC and ATP as a co-substrate monitored by LC-MS. Black lines indicate StaA prior to incubation with StaKC; red lines indicate 8-fold dehydrated product (1 h reaction time). Asterisks indicate a by-product from peptide synthesis. Figure 4. Schematic representation of StaA variant peptides (Thr→Ala, Ser→Ala and Cys→Ala substitutions) modified by StaKC. Number of IAA and βME adducts are listed (right side). Mutations at the C-terminus strongly interfere with maturation of StaA, whereas changes in N-terminal regions have a lesser influence on in vitro processing and cyclization. Asterisks show positions of βME-adducts and yions indicate fragmentation within the core peptide after MS/MS analysis of βME-derivatized peptides (Figure S9). Due to the lack of MS/MS data for peptide S3A/S6A a prediction of the ring structures is made based on the structures of other StaA-Ala-variants and on the number of βME adducts and lack of IAA adducts. Figure 5. Schematic presentation of biosynthetically flexible regions in the stackepeptin structure in vivo (A.) and in vitro (B.). A. Structures of the natural stackepeptins A, C and D. During in vivo maturation processing of the Ser15 and Ser26 by StaKC may be skipped rendering Lan rings. B. Products and ring topologies of the in vitro produced Ala-variant-StaA’s (red and green balls indicate positions of Ala exchanges). The lack of the C-terminal Ser hinders further full processing of the precursor peptide. Different N-terminal ring topologies are observed in vivo (Lab) and in vitro (Lan). 15 ACS Paragon Plus Environment


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Figure 1. A. Structural diversity of class III lanthipeptides. B. Biosynthetic gene cluster of stackepeptin with the sequence of the precursor peptide (recognition motif required for modification underlined, residues undergoing posttranslational modifications highlighted in orange). C. Alignment of type III lanthipeptide precursor peptides. Conserved amino acids are highlighted. 66x81mm (300 x 300 DPI)

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Figure 2. A. Proposed structures of stackepeptin A (1) and stackepeptin B (2). B. Structures of lanthipeptide characteristic amino acids labionin and lanthionine. C. LC-MS spectra of triply charged stackepeptins produced by S. nassauensis grown on two different media (upper panel – DSMZ medium No. 65, lower panel - same medium lacking CaCO3). D. Fragmentation patterns of 8-, 7- and 6-fold dehydrated natural stackepeptin A (1), C (3), and D (4) (y-ions are highlighted in blue color, b-ions are marked in red color; fragmentation spectra are presented in SI, Figure S3). 140x66mm (300 x 300 DPI)



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Figure 3. Scheme of the in vitro reaction of synthetic precursor peptide StaA with heterologously expressed His6-StaKC and ATP as a co-substrate monitored by LC-MS. Black lines indicate StaA prior to incubation with StaKC; red lines indicate 8-fold dehydrated product (1 h reaction time). Asterisks indicate a by-product from peptide synthesis. 66x64mm (300 x 300 DPI)


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Figure 4. Schematic representation of StaA variant peptides (Thr→Ala, Ser→Ala and Cys→Ala substitutions) modified by StaKC. Number of IAA and βME adducts are listed (right side). Mutations at the C-terminus strongly interfere with maturation of StaA, whereas changes in N-terminal regions have a lesser influence on in vitro processing and cyclization. Asterisks show positions of βME-adducts and y-ions indicate fragmentation within the core peptide after MS/MS analysis of βME-derivatized peptides (Figure S9). Due to the lack of MS/MS data for peptide S3A/S6A a prediction of the ring structures is made based on the structures of other StaA-Ala-variants and on the number of βME adducts and lack of IAA adducts. 67x84mm (300 x 300 DPI)



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Figure 5. Schematic presentation of biosynthetically flexible regions in the stackepeptin structure in vivo (A.) and in vitro (B.). A. Structures of the natural stackepeptins A, C and D. During in vivo maturation processing of the Ser15 and Ser26 by StaKC may be skipped rendering Lan rings. B. Products and ring topologies of the in vitro produced Ala-variant-StaA's (red and green balls indicate positions of Ala exchanges). The lack of the C-terminal Ser hinders further full processing of the precursor peptide. Different N-terminal ring topologies are observed in vivo (Lab) and in vitro (Lan). 140x63mm (300 x 300 DPI)


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Graphic for Table of contents 77x39mm (300 x 300 DPI)


The Supersized Class III Lanthipeptide Stackepeptin Displays Motif

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