Fabclavines: Bioactive Peptide–Polyketide-Polyamino Hybrids from Xenorhabdus Sebastian W. Fuchs,[a] Florian Grundmann,[a] Michael Kurz,[b] Marcel Kaiser,[c] and Helge B. Bode*[a] The structure of the fabclavines—unique mixtures of nonribosomally derived peptide–polyketide hybrids connected to an unusual polyamino moiety—has been solved by detailed NMR and MS methods. These compounds have been identified in two different entomopathogenic Xenorhabdus strains, thereby leading also to the identification of the fabclavine biosynthesis gene cluster. Detailed analysis of these clusters and initial mutagenesis experiments allowed the prediction of a biosynthesis pathway in which the polyamino moiety is derived from an unusual type of fatty acid synthase that is normally involved in formation of polyunsaturated fatty acids. As fabclavines show broad-spectrum activity against bacteria, fungi, and other eukaryotic cells, they might act as “protection factors” against all kinds of food competitors during the complex life cycle of Xenorhabdus, its nematode host, and their insect prey.

Here we describe the structure and possible biosynthesis of a novel class of hybrid peptide–polyketide-polyamino natural products: “fabclavines” (from Faber clavorum “nailsmith”, in memory of Willi Nagelschmidt) entomopathogenic Xenorhabdus strains.[6, 7] These exhibit broad-spectrum bioactivity against different organisms (Bacillus subtilis, Escherichia coli, Micrococcus luteus, Plasmodium falciparum, Saccharomyces cerevisiae, Trypanosoma brucei, Trypanosoma cruzi), and thus they might play a role in the complex life cycle of Xenorhabdus. This life cycle encompasses a symbiotic stage in the gut of their nematode hosts (Steinernema) and an entomopathogenic stage once the bacteria are released from the gut of their symbionts and enter the living insect body.[8] In their entomopathogenic stage, the bacteria kill the infected insect larvae and subsequently protect the dead prey from various food competitors, thereby allowing their own as well as their host’s reproduction.[9–11]

Introduction Natural products can be grouped, according to biochemical origin, as peptides, lipids, carbohydrates, alkaloids, or fatty acids (to name just a few important classes). As hybrids of these different classes also exist, the number of possible natural products awaiting identification (and possible application) is almost infinite. From the scientific point of view, natural products with unique structural features are of special interest, as these often reflect novel biochemistry or novel combinations of biochemical reactions. These reactions probably evolved millions of years ago; in this context, “novel” refers merely to the fact that they had not previously been recognized. Examples of such—and at the time, novel—biochemistry are hybrids of polyketide and nonribosomally derived peptides,[1, 2] compounds derived from trans-AT (acyltransferase) polyketide synthases (PKS),[3] and ribosomally derived peptides with heterocycles or even l- and d-amino acids.[4, 5] [a] S. W. Fuchs, F. Grundmann, Prof. Dr. H. B. Bode Merck Stiftungsprofessur fr Molekulare Biotechnologie Fachbereich Biowissenschaften Max-von-Laue-Strasse 9, 60438 Frankfurt am Main (Germany) E-mail: [email protected] [b] Dr. M. Kurz Sanofi–Aventis Deutschland GmbH (Industriepark Hoechst), Sanofi R&D Industriepark Hçchst, 65926 Frankfurt am Main (Germany) [c] M. Kaiser Swiss Tropical and Public Health Institute, Parasite Chemotherapy Socinstrasse 57, P. O. Box, 4002 Basel (Switzerland) Supporting information for this article is available on the WWW under

 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Results and Discussion Previous investigations of Xenorhabdus budapestensis indicated the presence of an unknown compound with high bioactivity against bacteria and oomycetes.[12, 13] MALDI-MS analysis of the culture supernatant from X. budapestensis yielded signals of unknown compounds, according to our in-house database of compounds specific to Xenorhabdus and Photorhabdus. Two sets of derivatives were identified, with differences of 10.01 and 44.02 Da (Figure S1 in the Supporting Information). Highresolution (HR)-MS and labeling experiments[14] indicated that the 10.01 Da difference arises from the exchange of a histidine to a phenylalanine moiety (Tables S1 and S2), and that the 44.02 Da difference between several derivatives correlates with addition or loss of C2H4O moieties, thus pointing to an at least partial polyketide nature of the compound (Table S1). The compounds from X. budapestensis were enriched as described previously.[10, 15] By using different steps of preparative chromatography (Chromatograms S1 and S2), one compound (1, 1356.96 Da) was purified. For NMR, several solvents were tried (DMSO, methanol, pyridine), but suitable spectra were obtained only when in water. To reduce line broadening of the amide resonances, NMR experiments were acquired at 286 K. The assignment of all proton and carbon resonances was carried out with various 2D NMR techniques, including DQF-COSY, TOCSY, ROESY, multiplicity edited-HSQC, HSQC-TOCSY and HMBC (Scheme 1, Table S3, Figures S2–S9). Two sets of signals were obtained, in a ratio of approximately 3.5:1 (see below). Primary analysis revealed the presence of five amino acids, ChemBioChem 2014, 15, 512 – 516



Scheme 1. Structure elucidation of fabclavine Ia (1) by 2D NMR experiments. Bonds shown in bold indicate spin systems which could be assigned by correlations in the DQF-COSY spectrum of 1. Sequential ROEs from the ROESY spectrum are indicated by black arrows. Because of line broadening only two sequential correlations could be obtained in the HMBC (significant HMBC correlations are indicated by broken arrows). For clarity only amide protons and signals from or to amide protons are shown. (Chemical shifts in Table S3; 1D and 2D NMR spectra in Figures S2–S9.)

including phenylalanine, 2,3-diaminobutyric acid (Dab), and Based on the structure of 1, the structures of three additionthree aspartic acid/aspartate residues. Additionally, a prolineal fabclavine derivatives (Table S1) produced by X. budapestenlike moiety was obtained, where the carbonyl function is resis were elucidated by MALDI-HCD-MS2 (Scheme 2, Figure S10 duced to a secondary alcohol. As indicated by correlations in and Table S4). The data confirmed that 2 (fabclavine Ib, the DQF-COSY spectrum, a C4-fragment is directly connected 1346.9 Da; Figure S10 B) is a derivative of 1 with a Phe-to-His to this proline-like moiety. Because of line broadening of most exchange at the 2-position. The MALDI-HCD-MS2 experiments amide resonances, sequential assignment was achieved mainly also proved that the 44.02 Da shift of derivatives of 1 and 2 through ROEs, and this led to the structure shown in results from only one (rather than two) CH2CHOH moiety at the C-terminal proline residue. Hence 3 (fabclavine IIa, Scheme 1. The terminal carboxyl function of the proline 1312.9 Da; Figure S10 C) and 4 (fabclavine Ib, 1302.9 Da; Figmoiety forms an amide bond with a primary amine that is part ure S10 D) are derivatives of 1 and 2, respectively, and harbor of a long linear carbon chain. Only the termini of this carbon only a single C2H4O extension of the proline residue. MALDIchain (C1–C6 and C33–C36) could be unambiguously assigned from the 2D spectra. For the central section, overlap of proton and carbon signals indicated the presence of repeating units of a methine group and two adjacent ( CH2 )n fragments (n  4). The carbon chemical shift of the methine group (52.16 ppm) indicates the presence of a directly connected amino function. From the results of the HRMS measurements and the integral values in the 1 H spectrum, the complete structure of the chain could be deduced. The second set of signals corresponded to a minor conformation of 1. Here, an intense ROE between Pro-Ha and DabHa proved a cis orientation of the amide bond between these two residues. An intense ROE between Pro-Hd and Dab-Ha was observed for the major conformation, thus indicating a trans orientation (Scheme 1). The compound, termed fabclavine Ia Scheme 2. Postulated structures of the fabclavines from A) X. budapestensis and B) X. szentirmaii identified in this (1), is similar to the recently de- work. The structure of 1 was determined by NMR experiments, flash Marfey’s analysis, and bioinformatics methods. The remaining fabclavines (2–5) were assumed to be derivatives of 1, and their structures were elucidated by scribed nemaucin,[16] but differs MALDI-HCD-MS2. The configurations of chiral centers were determined by the flash Marfey’s method, with predicby a C-terminal elongation of tion based on the presence of epimerization domains in the nonribosomal peptide synthetase or stereospecificity the peptide moiety. prediction of the ketoreductase domains.  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemBioChem 2014, 15, 512 – 516


CHEMBIOCHEM COMMUNICATIONS HCD-MS2 also afforded characterization of one of the fabclavines from Xenorhabdus szentirmaii, fabclavine IVa (5; Scheme 2, Figures S11–S13, Tables S5, S6). Structures of additional derivatives from X. szentirmaii were proposed based on their sum formulae (Figures S11–S13; Tables S5 and S6). In order to address the absolute configuration of the fabclavines, 1 was hydrolyzed and analyzed by the flash Marfey’s method (Table S7).[17] However, only the configuration of phenylalanine could be determined (unambigously as l; Figure S14). In order to predict the configuration of the other amino acids in the peptide moiety and the configuration of the hydroxy and amino moieties, the biosynthesis gene cluster of the fabclavines was identified and analyzed. Analysis of the genomes of X. budapestensis and X. szentirmaii by antiSMASH[18] resulted in the identification of a hybrid NRPS-PKS gene cluster (61 kb) that matched the expected NRPS-PKS module structure (in both genomes, with high similarity between each other). Bioinformatic analysis (Figure 1, Tables S8–S10),[19–21] allowed us to propose the mechanism of fabclavine biosynthesis (Figure 1 C). The involvement of the identified gene cluster in the biosynthesis of fabclavines was proved by mutagenesis of fclC and fclL in X. szentirmaii: this resulted in complete loss of fabclavine biosynthesis (Figure S15). Based on the BLASTP analysis of all proteins encoded by these clusters (Table S9), strong similarities with the zeamine biosynthesis gene clusters from Serratia plymuthica (and Dickeya

Figure 1. Biosynthesis gene cluster of fabclavines in the genomes of A) X. budapestensis and B) X. szentirmaii. Abbreviations for catalytic activities: Hy: hydrolase, KS: ketosynthase, AT: acyltransferase, T: thiolation domain, KR: ketoreductase, DH: dehydratase, Ox: 2-nitropropane dioxygenase (enoylreductase activity), AMT: aminotransferase, Red: thioester reductase, A: adenylation, C: condensation, E: epimerization, TP: transport, TR: transcription regulator, Pep: putative prodrug-peptidase, Nit: nitrilase. Solid triangles indicate the locations of the plasmid insertion sites (individually generated) in the gene cluster of X. szentirmaii DSM 16338. C) Proposed biosynthesis of 1 based on the observed composition and absolute configuration of fabclavines, the gene cluster architecture, and the observed phenotypes of the X. szentirmaii insertion mutants.

 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemBioChem 2014, 15, 512 – 516


CHEMBIOCHEM COMMUNICATIONS zeae) were observed (Figure S16).[22] Zeamine closely resembles the polyamino moiety of 1 (Scheme 1). In the case of S. plymuthica, the proposed pre-zeamine compound (Figure S16), whose structure would closely resemble the peptide and polyamino moiety of 1, was proposed to protect the producing organisms from potential lethality of the polyamino moiety. We postulate that the unusual polyamino moieties of the fabclavines are generated by the enzymes FclCDE, similarly to an earlier hypothesis.[22, 23] FclC and FclD are homologues of iterative type I PKSs or fatty acid synthases (FASs), similar to those for the biosynthesis of polyunsaturated fatty acids (PUFAs) found in many bacteria (Figure 1, Table S9).[22, 24] The aminotransferase domain (AMT) of FclE exhibits 24.4 % identity to the AMT involved in mycosubtilin biosynthesis,[25] and was predicted to be responsible for amine formation from 3-oxo acyl intermediates; the oxidase domain of FclE might act as PfaD-like enoylreductase, as described for PUFA biosynthesis.[24] The peptide moiety of the fabclavines is built by the NRPSs FclI and FclJ (Tables S8 and S9, Figure 1), with only one epimerization domain in the third module, specific for asparagine. This is in accordance with the results of the Marfey’s analysis (Table S7, Figure S14), which indicated the presence of one land one d-aspartic acid (derived from this asparagine during hydrolysis; Figure S14). Thus, the absolute configuration of the peptide part could be postulated (Scheme 2). The C-terminal elongation of the peptide moiety’s proline residue is catalyzed by the PKS FclK, which seems to catalyze the elongation with a maximum of two CH2CHOH functions. Peptide cyclization between the d-asparagine and the threonine residues might be catalyzed by FclH, which shows homology to nitrilases that have been described to catalyze reverse amidase reactions.[22, 26] A possible mechanism includes attack of the d-asparagine side chain’s amino function onto the b-carbon of the threonine residue, as well as possibly dehydration of the threonine side chain prior to the attack (Figure 1). In accordance with the proposed function of the C-domain-like protein FclL, the fclL insertion mutant exhibited a strong mass signal correlating to the expected mass of the polyamino moiety of the fabclavines from X. szentirmaii DSM 16338 (Figure S17). It was shown previously that the configuration of the secondary alcohol functions (as in the polyketide extension of the proline residue (positions 1’ and 3’) and in position 3 of the polyamino moiety of 1) can be reliably assigned based on bioinformatics analysis.[13, 27–29] Thus, based on a previous method,[30] the configurations of all these hydroxyl functions were assigned as R, by analyzing the primary structures of FclK, FclC, and FclF (Figure 1, Table S10). The configuration of the amino functions remains to be elucidated. Fabclavines Ia (1) and Ib (2) showed broad-spectrum activity against several organisms (Table S11). This broad activity points to general toxicity of fabclavines, which might act as “protection factors” against all sorts of food competitors in the soil (bacteria, fungi, protozoa) or in the gut of the insect prey (bacteria). This would facilitate establishment of a Xenorhabdus monoculture in the infected insect, thereby ensuring successful symbiosis between Xenorhabdus and its nematode host. Furthermore, fabclavines are structurally similar to cationic antimi 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim crobial peptides, which have been described to display pronounced synergistic effects in combination with other antibiotics.[31] This could even increase their bioactivity in vivo in combination with additional antibiotics in the cadaver; this is currently being tested by our group.

Acknowledgements We would like to thank Professor Michael Karas for his support and Peter Grn, Patrice Lubuta, and Hendrik Pollmann for technical assistance. This work was funded by the Deutsche Forschungsgemeinschaft (DFG) and in part by the European Community’s Seventh Framework Program (FP7/2007-2013) under grant agreement no. 602773. Keywords: biosynthesis · broad spectrum bioactivity · natural products · polyamino compounds · polyunsaturated fatty acid biosynthesis [1] B. Silakowski, H. U. Schairer, H. Ehret, B. Kunze, S. Weinig, G. Nordsiek, P. Brandt, H. Blçcker, G. Hçfle, S. Beyer, R. Mller, J. Biol. Chem. 1999, 274, 37391 – 37399. [2] B. Silakowski, G. Nordsiek, B. Kunze, H. Blçcker, R. Mller, Chem. Biol. 2001, 8, 59 – 69. [3] J. Piel, Nat. Prod. Rep. 2010, 27, 996 – 1047. [4] M. F. Freeman, C. Gurgui, M. J. Helf, B. I. Morinaka, A. R. Uria, N. J. Oldham, H.-G. Sahl, S. Matsunaga, J. Piel, Science 2012, 338, 387 – 390. [5] P. G. Arnison, M. J. Bibb, G. Bierbaum, A. A. Bowers, T. S. Bugni, G. Bulaj, J. A. Camarero, D. J. Campopiano, G. L. Challis, J. Clardy, P. D. Cotter, D. J. Craik, M. Dawson, E. Dittmann, S. Donadio, P. C. Dorrestein, K.-D. Entian, M. A. Fischbach, J. S. Garavelli, U. Gçransson, et al., Nat. Prod. Rep. 2013, 30, 108 – 160. [6] S. W. Fuchs, PhD thesis, Goethe Universitt Frankfurt am Main (Germany), 2013. [7] K. Lengyel, E. Lang, A. Fodor, E. Szlls, P. Schumann, E. Stackebrandt, Syst. Appl. Microbiol. 2005, 28, 115 – 122. [8] E. E. Herbert, H. Goodrich-Blair, Nat. Rev. Microbiol. 2007, 5, 634 – 646. [9] D. Reimer, K. M. Pos, M. Thines, P. Grn, H. B. Bode, Nat. Chem. Biol. 2011, 7, 888 – 890. [10] M. Gualtieri, A. Aumelas, J. O. Thaler, J. Antibiot. 2009, 62, 295 – 302. [11] H. B. Bode, Curr. Opin. Chem. Biol. 2009, 13, 224 – 230. [12] E. Bçszçrmnyi, T. rsek, A. Fodor, A. M. Fodor, L. S. Fçldes, M. Hevesi, J. S. Hogan, Z. Katona, M. G. Klein, A. Kormny, S. Pekr, A. Szentirmai, F. Sztaricskai, R. A. J. Taylor, J. Appl. Microbiol. 2009, 107, 746 – 759. [13] S. W. Fuchs, C. C. Sachs, C. Kegler, F. I. Nollmann, M. Karas, H. B. Bode, Anal. Chem. 2012, 84, 6948 – 6955. [14] H. B. Bode, D. Reimer, S. W. Fuchs, F. Kirchner, C. Dauth, C. Kegler, W. Lorenzen, A. O. Brachmann, P. Grn, Chem. Eur. J. 2012, 18, 2342 – 2348. [15] S. W. Fuchs, A. Proschak, T. W. Jaskolla, M. Karas, H. B. Bode, Org. Biomol. Chem. 2011, 9, 3130 – 3132. [16] A. Givaudan, M. Gualtieri, S. Pages, P. Villain-Guillot, PCT/EP2011/073738 [WO 2012085177 A1], 2013. [17] K. Fujii, Y. Yahashi, T. Nakano, S. Imanishi, S. F. Baldia, K.-i. Harada, Tetrahedron 2002, 58, 6873 – 6879. [18] M. H. Medema, K. Blin, P. Cimermancic, V. de Jager, P. Zakrzewski, M. A. Fischbach, T. Weber, E. Takano, R. Breitling, Nucleic Acids Res. 2011, 39, W339 – W346. [19] C. Rausch, T. Weber, O. Kohlbacher, W. Wohlleben, D. H. Huson, Nucleic Acids Res. 2005, 33, 5799 – 5808. [20] M. Rçttig, M. H. Medema, K. Blin, T. Weber, C. Rausch, O. Kohlbacher, Nucleic Acids Res. 2011, 39, W362 – W367. [21] B. O. Bachmann, J. Ravel, Methods Enzymol. 2009, 458, 181 – 217. [22] J. Masschelein, W. Mattheus, L.-J. Gao, P. Moons, R. Van Houdt, B. Uytterhoeven, C. Lamberigts, E. Lescrinier, J. Rozenski, P. Herdewijn, A. Aertsen, C. Michiels, R. Lavigne, PLoS One 2013, 8, e54143.

ChemBioChem 2014, 15, 512 – 516


CHEMBIOCHEM COMMUNICATIONS [23] J. Wu, H.-B. Zhang, J.-L. Xu, R. J. Cox, T. J. Simpson, L.-H. Zhang, Chem. Commun. 2010, 46, 333 – 335. [24] C. N. Shulse, E. E. Allen, PLoS One 2011, 6, e20146. [25] Z. D. Aron, P. C. Dorrestein, J. R. Blackhall, N. L. Kelleher, C. T. Walsh, J. Am. Chem. Soc. 2005, 127, 14986 – 14987. [26] H. C. Pace, C. Brenner, Genome Biol. 2001, 2, 0001. [27] C. J. Balibar, F. H. Vaillancourt, C. T. Walsh, Chem. Biol. 2005, 12, 1189 – 1200. [28] D. Reimer, N. C. Cowles, A. Proschak, F. I. Nollmann, A. J. Dowling, M. Kaiser, R. ffrench-Constant, H. Goodrich-Blair, H. B. Bode, ChemBioChem 2013, 14, 1991 – 1997.

 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim [29] A. Proschak, P. Lubuta, P. Grn, F. Lçhr, G. Wilharm, V. De Berardinis, H. B. Bode, ChemBioChem 2013, 14, 633 – 638. [30] A. Kitsche, M. Kalesse, ChemBioChem 2013, 14, 851 – 861. [31] R. E. W. Hancock, Lancet Infect. Dis. 2001, 1, 156 – 164.

Received: December 19, 2013 Published online on February 13, 2014

ChemBioChem 2014, 15, 512 – 516


Fabclavines: bioactive peptide-polyketide-polyamino hybrids from Xenorhabdus.

The structure of the fabclavines-unique mixtures of nonribosomally derived peptide-polyketide hybrids connected to an unusual polyamino moiety-has bee...
450KB Sizes 0 Downloads 3 Views