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Terrequinone A biosynthesis through L-tryptophan oxidation, dimerization and bisprenylation

Abstract

The antitumor fungal metabolite terrequinone A, identified in extracts of Aspergillus sp., is biosynthesized by the five-gene cluster tdiAtdiE. In this work, we have overproduced all five proteins (TdiA–TdiE) in the bacterial host Escherichia coli, fully reconstituting the biosynthesis of terrequinone A. This pathway involves aminotransferase activity, head-to-tail dimerization and bisprenylation of the scaffold to yield the benzoquinone natural product. We have established that TdiD is a pyridoxal-5′-phosphate–dependent L-tryptophan aminotransferase that generates indolepyruvate for an unusual nonoxidative coupling by the tridomain nonribosomal peptide synthetase TdiA. TdiC, an NADH-dependent quinone reductase, generates the nucleophilic hydroquinone for two distinct rounds of prenylation by the single prenyltransferase TdiB. TdiE is required to shunt the benzoquinone away from an off-pathway monoprenylated species by an as yet unknown mechanism. Overall, we have biochemically characterized the complete biosynthetic pathway to terrequinone A, highlighting the nonoxidative dimerization pathway and the unique asymmetric prenylation involved in its maturation.

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Figure 1: Structures of representative asterriquinones.
Figure 2: Formation of IPA from L-tryptophan by TdiD.
Figure 3: Characterization of TdiA.
Figure 4: Characterization of TdiB/TdiC/TdiE trienzyme system.
Figure 5: Turnover of intermediates on route to terrequinone A.
Figure 6: Biochemically characterized natural products derived from L-tryptophan dimerization.

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References

  1. Jerram, W.A. et al. The chemistry of cochliodinol, a metabolite of Chaetomium spp. Can. J. Chem. 53, 727–737 (1975).

    Article  CAS  Google Scholar 

  2. Meiler, D. & Taylor, A. The effect of cochliodinol, a metabolite of Chaetomium cochliodes, on the respiration of microsopores of Fusarium oxysporum. Can. J. Microbiol. 17, 83–86 (1971).

    Article  PubMed  CAS  Google Scholar 

  3. Brewer, D., Jerram, W.A. & Taylor, A. The production of cochliodinol and a related metabolite by Chaetomium species. Can. J. Microbiol. 14, 861–866 (1968).

    Article  PubMed  CAS  Google Scholar 

  4. Yamamoto, Y., Kiriyama, S., Shimizu, S. & Koshimura, S. Antitumor activity of asterrequinone, a metabolic product from Aspergillus terreus. Gann 67, 623–624 (1976).

    PubMed  CAS  Google Scholar 

  5. Arai, K. et al. Metabolic products of Aspergillus terreus. IV. Metabolite of the strain IFO 8835. (2). The isolation and structure of indolyl benzoquinone pigments. Chem. Pharm. Bull. (Tokyo) 29, 961–969 (1981).

    Article  CAS  Google Scholar 

  6. Mocek, U. et al. Isolation and structure elucidation of five new asterriquinones from Aspergillus, Humicola and Botryotrichum species. J. Antibiot. (Tokyo) 49, 854–859 (1996).

    Article  CAS  Google Scholar 

  7. Kaji, A., Saito, R., Nomura, M., Miyamoto, K. & Kiriyama, N. Mechanism of the cytotoxicity of asterriquinone, a metabolite of Aspergillus terreus. Anticancer Res. 17, 3675–3679 (1997).

    PubMed  CAS  Google Scholar 

  8. Shimizu, S., Yamamoto, Y., Inagaki, J. & Koshimura, S. Antitumor effect and structure-activity relationship of asterriquinone analogs. Gann 73, 642–648 (1982).

    PubMed  CAS  Google Scholar 

  9. He, J. et al. Cytotoxic and other metabolites of Aspergillus inhabiting the rhizosphere of Sonoran desert plants. J. Nat. Prod. 67, 1985–1991 (2004).

    Article  PubMed  CAS  Google Scholar 

  10. Bouhired, S., Weber, M., Kempf-Sontag, A., Keller, N.P. & Hoffmeister, D. Accurate prediction of the Aspergillus nidulans terrequinone gene cluster boundaries using the transcriptional regulator LaeA. Fungal Genet. Biol. (2007).

  11. Bok, J.W. et al. Genomic mining for Aspergillus natural products. Chem. Biol. 13, 31–37 (2006).

    Article  PubMed  CAS  Google Scholar 

  12. Marahiel, M.A., Stachelhaus, T. & Mootz, H.D. Modular peptide synthetases involved in nonribosomal peptide synthesis. Chem. Rev. 97, 2651–2674 (1997).

    Article  PubMed  CAS  Google Scholar 

  13. Stein, T. et al. The multiple carrier model of nonribosomal peptide biosynthesis at modular multienzymatic templates. J. Biol. Chem. 271, 15428–15435 (1996).

    Article  PubMed  CAS  Google Scholar 

  14. Belshaw, P.J., Walsh, C.T. & Stachelhaus, T. Aminoacyl-CoAs as probes of condensation domain selectivity in nonribosomal peptide synthesis. Science 284, 486–489 (1999).

    Article  PubMed  CAS  Google Scholar 

  15. Stachelhaus, T., Mootz, H.D., Bergendahl, V. & Marahiel, M.A. Peptide bond formation in nonribosomal peptide biosynthesis. Catalytic role of the condensation domain. J. Biol. Chem. 273, 22773–22781 (1998).

    Article  PubMed  CAS  Google Scholar 

  16. Conti, E., Stachelhaus, T., Marahiel, M.A. & Brick, P. Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S. EMBO J. 16, 4174–4183 (1997).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Stachelhaus, T., Huser, A. & Marahiel, M.A. Biochemical characterization of peptidyl carrier protein (PCP), the thiolation domain of multifunctional peptide synthetases. Chem. Biol. 3, 913–921 (1996).

    Article  PubMed  CAS  Google Scholar 

  18. Weber, T., Baumgartner, R., Renner, C., Marahiel, M.A. & Holak, T.A. Solution structure of PCP, a prototype for the peptidyl carrier domains of modular peptide synthetases. Structure 8, 407–418 (2000).

    Article  PubMed  CAS  Google Scholar 

  19. Keating, T.A. & Walsh, C.T. Initiation, elongation, and termination strategies in polyketide and polypeptide antibiotic biosynthesis. Curr. Opin. Chem. Biol. 3, 598–606 (1999).

    Article  PubMed  CAS  Google Scholar 

  20. Williams, R.M., Stocking, E.M. & Sanz-Cervera, J.F. Biosynthesis of prenylated alkaloids derived from tryptophan. Top. Curr. Chem. 209, 97–173 (2000).

    Article  CAS  Google Scholar 

  21. Dolence, J.M. & Poulter, C.D. in Comprehensive Natural Product Chemistry (ed. Meth-Cohn, O.) 18473–18500 (Elsevier, Oxford, 1999).

    Google Scholar 

  22. Grundmann, A. & Li, S.M. Overproduction, purification and characterization of FtmPT1, a brevianamide F prenyltransferase from Aspergillus fumigatus. Microbiology 151, 2199–2207 (2005).

    Article  PubMed  CAS  Google Scholar 

  23. Eliot, A.C. & Kirsch, J.F. Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations. Annu. Rev. Biochem. 73, 383–415 (2004).

    Article  PubMed  CAS  Google Scholar 

  24. Magarvey, N.A., Ehling-Schulz, M. & Walsh, C.T. Characterization of the cereulide NRPS alpha-hydroxy acid specifying modules: activation of alpha-keto acids and chiral reduction on the assembly line. J. Am. Chem. Soc. 128, 10698–10699 (2006).

    Article  PubMed  CAS  Google Scholar 

  25. Quadri, L.E. et al. Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases. Biochemistry 37, 1585–1595 (1998).

    Article  PubMed  CAS  Google Scholar 

  26. Nishizawa, T., Aldrich, C.C. & Sherman, D.H. Molecular analysis of the rebeccamycin L-amino acid oxidase from Lechevalieria aerocolonigenes ATCC 39243. J. Bacteriol. 187, 2084–2092 (2005).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Gebler, J.C. & Poulter, C.D. Purification and characterization of dimethylallyl tryptophan synthase from Claviceps purpurea. Arch. Biochem. Biophys. 296, 308–313 (1992).

    Article  PubMed  CAS  Google Scholar 

  28. Lee, S.L., Floss, H.G. & Heinstein, P. Purification and properties of dimethylallylpyrophosphate:tryptopharm dimethylallyl transferase, the first enzyme of ergot alkaloid biosynthesis in Claviceps. sp. SD 58. Arch. Biochem. Biophys. 177, 84–94 (1976).

    Article  PubMed  CAS  Google Scholar 

  29. Unsold, I.A. & Li, S.M. Overproduction, purification and characterization of FgaPT2, a dimethylallyltryptophan synthase from Aspergillus fumigatus. Microbiology 151, 1499–1505 (2005).

    Article  PubMed  CAS  Google Scholar 

  30. Hamahata, A., Takata, Y., Gomi, T. & Fujioka, M. Probing the S-adenosylmethionine-binding site of rat guanidinoacetate methyltransferase. Effect of site-directed mutagenesis of residues that are conserved across mammalian non-nucleic acid methyltransferases. Biochem. J. 317, 141–145 (1996).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. de Guzman, F.S. et al. Ochrindoles A-D: new bis-indolyl benzenoids from the sclerotia of Aspergillus ochraceus NRRL 3519. J. Nat. Prod. 57, 634–639 (1994).

    Article  PubMed  CAS  Google Scholar 

  32. Howard-Jones, A.R. & Walsh, C.T. Enzymatic generation of the chromopyrrolic acid scaffold of rebeccamycin by the tandem action of RebO and RebD. Biochemistry 44, 15652–15663 (2005).

    Article  PubMed  CAS  Google Scholar 

  33. Onaka, H., Taniguchi, S., Igarashi, Y. & Furumai, T. Characterization of the biosynthetic gene cluster of rebeccamycin from Lechevalieria aerocolonigenes ATCC 39243. Biosci. Biotechnol. Biochem. 67, 127–138 (2003).

    Article  PubMed  CAS  Google Scholar 

  34. Sanchez, C. et al. The biosynthetic gene cluster for the antitumor rebeccamycin: characterization and generation of indolocarbazole derivatives. Chem. Biol. 9, 519–531 (2002).

    Article  PubMed  CAS  Google Scholar 

  35. Asamizu, S., Kato, Y., Igarashi, Y., Furumai, T. & Onaka, H. Direct formation of chromopyrrolic acid from indole-3-pyruvic acid by StaD, a novel hemoprotein in indolocarbazole biosynthesis. Tetrahedr. Lett. 47, 473–475 (2006).

    Article  CAS  Google Scholar 

  36. Howard-Jones, A.R. & Walsh, C.T. Staurosporine and rebeccamycin aglycones are assembled by the oxidative action of StaP, StaC, and RebC on chromopyrrolic acid. J. Am. Chem. Soc. 128, 12289–12298 (2006).

    Article  PubMed  CAS  Google Scholar 

  37. Onaka, H., Taniguchi, S., Igarashi, Y. & Furumai, T. Cloning of the staurosporine biosynthetic gene cluster from Streptomyces sp. TP-A0274 and its heterologous expression in Streptomyces lividans. J. Antibiot. (Tokyo) 55, 1063–1071 (2002).

    Article  CAS  Google Scholar 

  38. Balibar, C.J. & Walsh, C.T. In vitro biosynthesis of violacein from L-tryptophan by the enzymes VioA-E from Chromobacterium violaceum. Biochemistry 45, 15444–15457 (2006).

    Article  PubMed  CAS  Google Scholar 

  39. Hibino, S. & Choshi, T. Simple indole alkaloids and those with a nonrearranged monoterpenoid unit. Nat. Prod. Rep. 19, 148–180 (2002).

    Article  PubMed  CAS  Google Scholar 

  40. Sanchez, C., Mendez, C. & Salas, J.A. Indolocarbazole natural products: occurrence, biosynthesis, and biological activity. Nat. Prod. Rep. 23, 1007–1045 (2006).

    Article  PubMed  CAS  Google Scholar 

  41. Bao, B. et al. Bisindole alkaloids of the topsentin and hamacanthin classes from a marine sponge Spongosorites sp. J. Nat. Prod. 70, 2–8 (2007).

    Article  PubMed  CAS  Google Scholar 

  42. Oh, K.B. et al. Bis(indole) alkaloids as sortase A inhibitors from the sponge Spongosorites sp. Bioorg. Med. Chem. Lett. 15, 4927–4931 (2005).

    Article  PubMed  CAS  Google Scholar 

  43. Endo, T., Tsuda, M., Fromont, J. & Kobayashi, J. Hyrtinadine A, a bis-indole alkaloid from a marine sponge. J. Nat. Prod. 70, 423–424 (2007).

    Article  PubMed  CAS  Google Scholar 

  44. Nishizawa, T., Gruschow, S., Jayamaha, D.H., Nishizawa-Harada, C. & Sherman, D.H. Enzymatic assembly of the bis-indole core of rebeccamycin. J. Am. Chem. Soc. 128, 724–725 (2006).

    Article  PubMed  CAS  Google Scholar 

  45. Unsold, I.A. & Li, S.M. Reverse prenyltransferase in the biosynthesis of fumigaclavine C in Aspergillus fumigatus: gene expression, purification, and characterization of fumigaclavine C synthase FGAPT1. ChemBioChem 7, 158–164 (2006).

    Article  PubMed  CAS  Google Scholar 

  46. Stachelhaus, T., Mootz, H.D. & Marahiel, M.A. The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem. Biol. 6, 493–505 (1999).

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We gratefully acknowledge the National Institutes of Health grant GM 20011 (to C.T.W.) and a Department of Defense National Defense Science and Engineering Graduate Fellowship (to C.J.B.). We thank P.D. Straight for discussions and for providing a sample of A. nidulans strain A4, and we thank J.A. Read for his critical reading of this manuscript.

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C.J.B. and A.R.H.-J. contributed equally to this work.

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Correspondence to Christopher T Walsh.

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Balibar, C., Howard-Jones, A. & Walsh, C. Terrequinone A biosynthesis through L-tryptophan oxidation, dimerization and bisprenylation. Nat Chem Biol 3, 584–592 (2007). https://doi.org/10.1038/nchembio.2007.20

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