Acknowledgements This work was supported by funding of the Academy of Finland (259505), Helsinki University Research grant (490085) and ESCMID grant (4720572) to D.P.F., University of Pittsburgh Central Research Development Fund to X.L., Technology Strategy Board grant (131181) to W.H., M.J. and J.H.N. National Programme of Sustainability I of the Ministry of Education of the Czech Republic I grant (LO1416) to T.G.Peer reviewedPostprin

Arnison

Bent

Donia

Houssen

Hulvová

Ishida

Koehnke

Leikoski

Magnuson

Martin

McIntosh

Shanks

Sivonen

Tsuji

Wallwey

Williams

Angewandte Chemie International Edition

English

PubMed

Cyanobactins are a rapidly growing family of linear and cyclic peptides produced by cyanobacteria. Kawaguchipeptins A and B, two macrocyclic undecapeptides reported earlier from Microcystis aeruginosa NIES-88, are shown to be products of the cyanobactin biosynthetic pathway. The 9 kb kawaguchipeptin (kgp) gene cluster was identified in a 5.26 Mb draft genome of Microcystis aeruginosa NIES-88. We verified that this gene cluster is responsible for the production of the kawaguchipeptins through heterologous expression of the kgp gene cluster in Escherichia coli. The KgpF prenyltransferase was overexpressed and was shown to prenylate C-3 of Trp residues in both linear and cyclic peptides in vitro. Our findings serve to further enhance the structural diversity of cyanobactins to include tryptophan-prenylated cyclic peptides

Parajuli, Anirudra

Kwak, Daniel H.

Dalponte, Luca

Leikoski, Niina

Galica, Tomas

Umeobika, Ugochukwu

Trembleau, Laurent

Bent, Andrew

Sivonen, Kaarina

Wahlsten, Matti

Wang, Hao

Rizzi, Ermanno

De Bellis, Gianluca

Naismith, Jim

Jaspars, Marcel

Liu, Xinyu

Houssen, Wael

Fewer, David Peter

University of St. Andrews - Pure

A unique Tryptophan C-prenyltransferase from the kawaguchipeptin biosynthetic pathway

Anirudra Parajuli

Daniel H. Kwak

Luca Dalponte

Niina Leikoski

Tomas Galica

Ugochukwu Umeobika

Laurent Trembleau

Andrew Bent

Kaarina Sivonen

Matti Wahlsten

Hao Wang

Ermanno Rizzi

Gianluca De Bellis

James Naismith

Marcel Jaspars

Xinyu Liu

Wael Houssen

David Peter Fewer

Crossref

Angewandte Chemie

A Unique Tryptophan C-Prenyltransferase from the Kawaguchipeptin Biosynthetic Pathway

University of St Andrews Research Portal

Acknowledgements This work was supported by funding of the Academy of Finland (259505), Helsinki University Research grant (490085) and ESCMID grant (4720572) to D.P.F., University of Pittsburgh Central Research Development Fund to X.L., Technology Strategy Board grant (131181) to W.H., M.J. and J.H.N. National Programme of Sustainability I of the Ministry of Education of the Czech Republic I grant (LO1416) to T.G.Peer reviewe

Rizzi, Ermano

De Bellis, Gianluca

Naismith, James

Aberdeen University Research

German Edition: DOI: 10.1002/ange.201509920BiosynthesisInternational Edition: DOI: 10.1002/anie.201509920AUnique Tryptophan C-Prenyltransferase from the KawaguchipeptinBiosynthetic PathwayAnirudra Parajuli+, Daniel H. Kwak+, Luca Dalponte+, Niina Leikoski, Tomas Galica,Ugochukwu Umeobika, Laurent Trembleau, Andrew Bent, Kaarina Sivonen, Matti Wahlsten,Hao Wang, Ermanno Rizzi, Gianluca De Bellis, James Naismith, Marcel Jaspars, Xinyu Liu,*Wael Houssen,* and David Peter Fewer*Abstract: Cyanobactins are a rapidly growing family of linearand cyclic peptides produced by cyanobacteria. Kawaguchi-peptins A and B, two macrocyclic undecapeptides reportedearlier from Microcystis aeruginosa NIES-88, are shown to beproducts of the cyanobactin biosynthetic pathway. The 9 kbkawaguchipeptin (kgp) gene cluster was identified in a 5.26 Mbdraft genome of Microcystis aeruginosa NIES-88. We verifiedthat this gene cluster is responsible for the production of thekawaguchipeptins through heterologous expression of the kgpgene cluster in Escherichia coli. The KgpF prenyltransferasewas overexpressed and was shown to prenylate C-3 of Trpresidues in both linear and cyclic peptides in vitro. Our findingsserve to further enhance the structural diversity of cyanobactinsto include tryptophan-prenylated cyclic peptides.Cyanobactins are a family of ribosomally-synthesized andposttranslationally modified peptides (RiPPs) produced bycyanobacteria.[1–6] Cyanobactin posttranslational modifica-tions include N-to-C macrocyclization; epimerization; heter-ocyclization to form thiazolines and oxazolines; oxidation ofheterocycles to thiazoles and oxazoles; N-methylation of His;O-prenylation on Ser, Thr, and Tyr; and N-prenylation.[2,7,8]Biological activities that have been reported for cyanobactinsinclude anticancer, antimalarial, antibacterial, and proteaseinhibitory activity.[2]The posttranslational prenylation of cyanobactins iscatalyzed by a prenyltransferase enzyme encoded within thecyanobactin biosynthetic gene cluster, which uses 3-methyl-but-2-en-1-yl group derived from dimethylallyl pyrophos-phate (DMAPP).[9–12] Although a putative prenyltransferasegene is present in all known cyanobactin gene clusters, onlya few of the cyanobactins are known to be prenylated,including prenylagaramides, aestuaramides, trunkamides, andanacyclamides.[4,6, 10,12] The known cyanobactin prenyltrans-ferases are O-prenyltransferases that catalyze the O-prenyla-tion of Tyr, Thr, and Ser in forward or reverse orientation. C-prenylated cyanobactins have been shown to be synthesizedoriginally as O-prenylated peptides that later undergoa Claisen rearrangement to yield C-prenylated peptides.[12]Kawaguchipeptins are macrocyclic undecapeptides pro-duced by the cyanobacterial strain Microcystis aeruginosaNIES-88.[13,14] Two variants of kawaguchipeptin have beenreported (Scheme 1). Kawaguchipeptin A contains two C-3-prenylated tryptophan residues and a d-Leu residue.[13]Kawaguchipeptin B consists of solely unmodified aminoacids and is reported to show antimicrobial activity againstStaphylococcus aureus.[14] Herein, we report a genomesequence for Microcystis aeruginosa NIES-88, identify thekawaguchipeptin biosynthetic gene cluster, and confirmenzymatic prenylation activity.[*] A. Parajuli,[+] Dr. N. Leikoski, T. Galica, Prof. K. Sivonen, M. Wahlsten,Dr. H. Wang, Dr. D. P. FewerMicrobiology and Biotechnology DivisionDepartment of Food and Environmental SciencesP.O.Box 56, Viikki Biocenter, Viikinkaari 9, 00014University of Helsinki (Finland)E-mail: david.fewer@helsinki.fiD. H. Kwak,[+] Prof. X. LiuDepartment of Chemistry, University of Pittsburgh219 Parkman Avenue, Pittsburgh, PA 15260 (USA)E-mail: xinyuliu@pitt.eduL. Dalponte,[+] U. Umeobika, Dr. L. Trembleau, Prof. M. Jaspars,Dr. W. HoussenMarine Biodiscovery Centre, Department of ChemistryUniversity of Aberdeen, Meston Walk, Aberdeen AB24 3UE (UK)E-mail: w.houssen@abdn.ac.ukL. Dalponte,[+] Dr. W. HoussenInstitute of Medical Sciences, University of AberdeenAberdeen AB25 2ZD (UK)T. GalicaInstitute of Microbiology AS CR, v.v.i. , Center ALGATECH, Trˇebonˇ(Czech Republic)andUniversity of South Bohemia, Faculty of ScienceDepartment of Ecosystem BiologyCˇesk Budeˇjovice (Czech Republic)A. Bent, Prof. J. NaismithBiomedical Sciences Research Complex, University of St AndrewsNorth Haugh, St Andrews, Fife KY16 9ST (UK)Dr. E. Rizzi, Prof. G. De BellisInstitute for Biomedical Technologies (ITB)National Research Council (CNR)via F.lli Cervi 93, Segrate (MI) (Italy)Dr. W. HoussenPharmacognosy Department, Faculty of PharmacyMansoura University, Mansoura 35516 (Egypt)[+] These authors contributed equally to this work.Supporting information for this article (including experimentaldetails) can be found under http://dx.doi.org/10.1002/anie.201509920.AngewandteChemieCommunications1Angew. Chem. Int. Ed. 2016, 55, 1 – 5  2016 Wiley-VCH Verlag GmbH & Co. KGaA, WeinheimThese are not the final page numbers! We obtained a 5.26 Mb genome sequence for Microcystisaeruginosa NIES-88 by using a combination of 3 kb 454sequencing and short-insert illumina Miseq paired-end data,which were subsequently assembled into 29 scaffolds. A totalof 4,996 genes, including 4 ribosomal RNA operons and41 tRNAs, were annotated from this genome. The kawagu-chipeptin precursor gene was identified through tBLASTnusing the predicted kawaguchipeptin peptide backbone(WLNGDNNWSTP). The KgpE precursor peptide was found toencode three exact copies of the WLNGDNNWSTP core(Figure 1). The KgpE precursor peptide was encoded ina 9 kb gene cluster (kgp) together with the KgpA and KgpGcyanobactin proteases, as well as the putative KgpF prenyl-transferase (Figure 1). The KgpF prenyltransferase shows just20–45% identity to known and putative cyanobactin prenyl-transferases. A homologue of cyclodehydrase (PatD) was notdetected in the gene cluster, which is consistent with theabsence of heterocyclized amino acids in the cyclic peptides.To demonstrate that the kgpA-G genes encode kawagu-chipeptin production in vivo, we cloned the entire kgp operoninto a broad host range yeast/bacteria shuttle vectorpMQ123i[15] and placed kgpA downstream of a pTac pro-moter to generate the expression plasmid pDK-kgp1 (Fig-ure S1 in the Supporting Information). This construct allowedthe regulated expression of the kgpA-G genes in E. coliTOP10. Liquid chromatography with high-resolution massspectrometry (LC–HRMS) guided metabolite profiling of E.coli cells transformed with pDK-kgp1 revealed the presenceof 2, for which the LC retention time and HRMS profilematched authentic 2 isolated from M. aeruginosa NIES-88(Figure 2, traces 2–3, and Figure S2). E. coli cells carryingpDK-kgp1 alone, however, did not produce 1 (Figure 2,trace 5), which is potentially derived from 2 by bisprenylationat C-3 of Trp by prenyltransferase KgpF. We hypothesizedthat this observation may have been due to the lack ofsufficient endogenous prenyl donor dimethylallyl pyrophos-phate (DMAPP) in E. coli. To overcome this problem, we co-transformed E. coli with pDK-kgp1 and the plasmid pMBI,[16]which harbors four yeast mevalonate-dependent isoprenoidpathway biosynthetic genes that can convert mevalonate toisopentenyl pyrophosphate (IPP), a precursor to DMAPP.Co-expression of the pDK-kgp1 and pMBI genes in E. coliTOP10 supplied with exogenous mevalonolactone 3 (1.0 mm)led to the production of both 1 and 2 that matched theirauthentic standards from M. aeruginosa NIES-88 (Figure 2,traces 1–2,6–7, and Figure S3), as assessed by LC–HRMS.These experiments establish that the kgpA-G genes conferScheme 1. The cyclic undecapeptides kawaguchipeptin A (1) and kawa-guchipeptin B (2) reported from Microcystis aeruginosa NIES-88. Kawa-guchipeptin A contains two C-3-prenylated tryptophan residues (high-lighted). Prenylation of C-3 is coupled to the formation of a new ringformation as a result of bond formation between C-2 and NH in themain chain.Figure 1. The kawaguchipeptin biosynthetic pathway in Microcystisaeruginosa NIES-88.[23] A) The 9 kb kgp biosynthetic gene clusterconsists of six biosynthetic genes (shown in blue), organized ina single operon, that show homology to genes present in other knowncyanobactin gene clusters. The grey arrows indicate genes that encodeproteins with unknown functions. B) The kgpE precursor gene encodesthe 87 amino acid precursor peptide, which contains three identicalcopies of the undecapeptide core (highlighted in grey).Figure 2. Heterologous expression of the kgp operon in E. coli demon-strates that the kgpA-G genes confer the production of 1 and 2 in vivo.Extracted ion chromatographs of LC–HRMS analysis of: Authentic1 and 2 from M. aeruginosa NIES-88 (1,2); 2 in E. coli transformedwith pDK-kgp1 (3); 1 in E. coli transformed with pDK-kgp1 (4); 1 in E.coli co-transformed with pDK-kgp1 and pMBI and supplied withmevalonolactone 3 (5); and 2 in E. coli co-transformed with pDK-kgp1and pMBI and supplied with mevalonolactone 3 (6).AngewandteChemieCommunications2 www.angewandte.org  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2016, 55, 1 – 5These are not the final page numbers!1 and 2 biogenesis in vivo. In addition, the coexistence of1 and 2 in E. coli cells transformed with both pDK-kgp1 andpMBI and the lack of 1 in E. coli cells transformed with pDK-kgp1 alone strongly suggest that 2 is the direct biosyntheticprecursor to 1 and bis-prenylation by KgpF is likely the lastenzymatic step in the biosynthetic maturation of kawaguchi-peptins.We overexpressed and purified the recombinant KgpFfrom E. coli and assessed the enzymes ability to processa range of cyclic and linear peptides and to use isopentenylpyrophosphate (IPP), DMAPP, and geranyl pyrophosphate(GPP; Table 1 and Figures S4–S24).Our results show that the enzyme processes a second Trpresidue within macrocyclic peptide substrate 2, whereas in thelinear peptide 9, despite 40 h of incubation, only a singlemodification was observed with DMAPP as the cofactor(Table 1; Figures S5–S7,S18–S20). The catalytic activity of theenzyme decreased when IPP was used as a cofactor instead ofDMAPP, as seen in the processing of one Trp residue out oftwo in kawaguchipeptin B (2) when IPP instead of DMAPPwas used (Table 1; Figures S8). We did not detect anyprocessing of other residues in the linear or macrocyclicpeptide substrates. The enzyme did not process Boc-Trp (13)in presence of DMAPP or IPP. Interestingly, the enzymecould not use GPP as a cofactor. According to these results,the selectivity of prenylation in linear substrates can beexplained by the necessity of the Trp residue to be sandwichedbetween two residues. Terminal Trp residues would presum-ably be too strongly solvated to bind efficiently the enzymaticsite and undergo transformation (Figures S20,S22).O- or C-prenylation of Tyr, Ser, and Thr in forward orreverse orientation have been observed for cyclic cyanobac-tins.[2,5, 12] C-prenylated peptides like the C-prenylated Tyr inaestuaramides have been reported.[12] However, the latter isthe result of reverse O-prenylation on the oxygen atom of Tyrfollowed by Claisen rearrangement.[12] The biochemicalcharacterization of C-3 Trp prenylation[13] and demonstrationof the presence of a homologue of the prenyltransferase genekgpF in the gene cluster confirm that this is a directposttranslational modification, which is rare in cyanobactins.To our knowledge, C-3 prenylation of Trp through posttrans-lational modification of a peptide has been demonstrated onlyonce before for the ComX peptide, a pheromone produced byBacillus subtilis and related bacilli.[17,18]Trp prenylation is common in some plants and bacteria,but mostly in fungi, and the respective prenyltransferasescatalyze the addition of a dimethylallyl group to Trp duringthe synthesis of secondary metabolites.[19,20,21] The synthesisand biosynthesis of these compounds, particularly indolealkaloids that contain prenylated Trp at their core, have beenthe subject of considerable interest.[22] Our findings thereforeexpand the chemical diversity of cyanobactins and confirmthe existence of a rare Trp prenyltransferase. The prenyl-transferases of the cyanobactin family are now known tocatalyze the O-, C-, and N-prenylation of amino acids in cyclicpeptides.AcknowledgementsThis work was supported by funding of the Academy ofFinland (259505), Helsinki University Research grant(490085) and ESCMID grant (4720572) to D.P.F., Universityof Pittsburgh Central Research Development Fund to X.L.,Technology Strategy Board grant (131181) to W.H., M.J. andJ.H.N. National Programme of Sustainability I of the Ministryof Education of the Czech Republic I grant (LO1416) to T.G.Keywords: biosynthesis · cyanobactins · peptides · prenylation ·prenyltransferases[1] P. G. Arnison, et al., Nat. Prod. Rep. 2013, 30, 108 – 160.[2] K. Sivonen, N. Leikoski, D. P. Fewer, J. Jokela, Appl. Microbiol.Biotechnol. 2010, 86, 1213 – 1225.[3] N. Leikoski, D. P. Fewer, K. Sivonen, Appl. Environ. Microbiol.2009, 75, 853 – 857.[4] N. Leikoski, D. P. Fewer, J. Jokela, M. Wahlsten, L. Rouhiainen,K. Sivonen, Appl. Environ. Microbiol. 2010, 76, 701 – 709.[5] N. Leikoski, L. Liu, J. Jokela, M. Wahlsten, M. Gugger, A.Calteau, P. Permi, C. A. Kerfeld, K. Sivonen, D. P. Fewer, Chem.Biol. 2013, 20, 1033 – 1043.[6] M. S. Donia, E. W. Schmidt, Chem. Biol. 2011, 18, 508 – 519.[7] J. Koehnke, et al., Angew. Chem. Int. Ed. 2013, 52, 13991 –13996; Angew. Chem. 2013, 125, 14241 – 14246.[8] W. E. Houssen, et al., Angew. Chem. Int. Ed. 2014, 53, 14171 –14174; Angew. Chem. 2014, 126, 14395 – 14398.[9] A. F. Bent, J. Koehnke, W. E. Houssen, M. C. Smith, M. Jaspars,J. M. Naismith, Acta Crystallogr. Sect. F 2013, 69, 618 – 623.[10] M. S. Donia, B. J. Hathaway, S. Sudek, M. G. Haygood, M.Rosovitz, J. Ravel, E. W. Schmidt,Nat. Chem. Biol. 2006, 2, 729 –735.[11] M. S. Donia, J. Ravel, E. W. Schmidt, Nat. Chem. Biol. 2008, 4,341 – 343.[12] J. A. McIntosh, M. S. Donia, S. K. Nair, E. W. Schmidt, J. Am.Chem. Soc. 2011, 133, 13698 – 13705.Table 1: Substrates and cofactors used for the in vitro reactions.Substrate Cofactor Mono-prenylationDiprenylationCyclic [WLNGDNNWSTP] (2) DMAPP + +Cyclic [WLNGDNNWSTP] (2) IPP + Cyclic [WLNGDNNWSTP] (2) GPP  Cyclic [TSQIWGSPVP] (4) DMAPP + NACyclic [SAQWQNFGVP] (5) DMAPP + NACyclic [HAFIGYDQDPTGKYP] (6) DMAPP  Cyclic [RERFVYP] (7) DMAPP  Cyclic [LIGIMHP] (8) DMAPP  WLNGDNNWSTP (9) DMAPP + WLNGDNNWSTPAYDG (10) DMAPP + EDWYFDHPAYDG (11) DMAPP  VPWPFPAYDG (12) DMAPP  Boc-Trp (13) DMAPP  Boc-Trp (13) IPP  Boc-Tyr (14) DMAPP  Boc-Tyr (14) IPP  [+] Product detected, [] Product not detected.AngewandteChemieCommunications3Angew. Chem. Int. Ed. 2016, 55, 1 – 5  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.orgThese are not the final page numbers! [13] K. Ishida, H. Matsuda, M. Murakami, K. Yamaguchi, Tetrahe-dron 1996, 52, 9025 – 9030.[14] K. Ishida, H. Matsuda, M. Murakami, K. Yamaguchi, J. Nat.Prod. 1997, 60, 724 – 726.[15] R. M. Q. Shanks, D. E. Kadouri, D. P. MacEachran, G. OToole,Plasmid 2009, 62, 88 – 97.[16] V. J. Martin, D. J. Pitera, S. T. Withers, J. D. Newman, J. D.Keasling, Nat. Biotechnol. 2003, 21, 796 – 802.[17] R. Magnuson, J. Solomon, A. D. Grossman, Cell 1994, 77, 207 –216.[18] F. Tsuji, K. Kobayashi, M. Okada, H. Yamaguchi, M. Ojika, Y.Sakagami, Bioorg. Med. Chem. Lett. 2011, 21, 4041 – 4044.[19] S. Li, Nat. Prod. Rep. 2010, 27, 57 – 78.[20] C. Wallwey, S. Li, Nat. Prod. Rep. 2011, 28, 496 – 510.[21] H. Hulvov, P. Galuszka, J. Frbortov, I. Frbort, Biotechnol.Adv. 2013, 31, 79 – 89.[22] R. M. Williams, E. M. Stocking, J. F. Sanz-Cervera, Top. Curr.Chem. 2000, 209, 97 – 173.[23] The draft genome of Microcystis aeruginosa NIES-88 has beendeposited in GenBank under the accession numberJXYX00000000.Received: October 23, 2015Published online: && &&, &&&&AngewandteChemieCommunications4 www.angewandte.org  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2016, 55, 1 – 5These are not the final page numbers!CommunicationsBiosynthesisA. Parajuli, D. H. Kwak, L. Dalponte,N. Leikoski, T. Galica, U. Umeobika,L. Trembleau, A. Bent, K. Sivonen,M. Wahlsten, H. Wang, E. Rizzi,G. De Bellis, J. Naismith, M. Jaspars,X. Liu,* W. Houssen,*D. P. Fewer* &&&&—&&&&A Unique Tryptophan C-Prenyltransferasefrom the Kawaguchipeptin BiosyntheticPathwayThe macrocyclic undecapeptides Kawa-guchipeptin A and B from Microcystisaeruginosa NIES-88 were shown to beproducts of the cyanobactin biosyntheticpathway. The 9 kb kawaguchipeptin (kgp)gene cluster was identified as beingresponsible for their production throughheterologous expression in Escherichiacoli, and the KgpF prenyltransferase wasshown to prenylate C-3 of Trp residues inboth linear and cyclic peptides in vitro.AngewandteChemieCommunications5Angew. Chem. Int. Ed. 2016, 55, 1 – 5  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.orgThese are not the final page numbers! 

A Unique Tryptophan C‐Prenyltransferase from the Kawaguchipeptin Biosynthetic Pathway

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A Unique Tryptophan C‐Prenyltransferase from the Kawaguchipeptin Biosynthetic Pathway

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