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Molecular basis for the P450-catalyzed C–N bond formation in indolactam biosynthesis

Nature Chemical Biology volume 15, pages 1206–1213 (2019)Cite this article

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Abstract

The catalytic versatility of cytochrome P450 monooxygenases is remarkable. Here, we present mechanistic and structural characterizations of TleB from Streptomyces blastmyceticus and its homolog HinD from Streptoalloteichus hindustanus, which catalyze unusual intramolecular C–N bond formation to generate indolactam V from the dipeptide N-methylvalyl-tryptophanol. In vitro analyses demonstrated that both P450s exhibit promiscuous substrate specificity, and modification of the N13-methyl group resulted in the formation of indole-fused 6/5/6 tricyclic products. Furthermore, X-ray crystal structures in complex with substrates and structure-based mutagenesis revealed the intimate structural details of the enzyme reactions. We propose that the generation of a diradical species is critical for the indolactam formation, and that the intramolecular C(sp2)–H amination is initiated by the abstraction of the N1 indole hydrogen. After indole radical repositioning and subsequent removal of the N13 hydrogen, the coupling of the properly-folded diradical leads to the formation of the C4–N13 bond of indolactam.

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Fig. 1: Biosynthetic pathway of indolactams.
Fig. 2: Enzyme reactions of TleB and HinD.
Fig. 3: Enzyme reactions of TleB and HinD with various substrate analogs.
Fig. 4: The binding modes of substrates in the active sites of TleB and HinD.
Fig. 5: Enzyme reactions of TleB and HinD mutants.
Fig. 6: Proposed mechanism for the TleB and HinD enzymatic reaction.

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Data availability

The coordinates and structure factor amplitudes for the apo structures of TleB and HinD and those complexed with ligands were deposited to the Protein Data Bank (PDB) under accession codes 6J82, 6J83, 6J84, 6J85, 6J86, 6J87 and 6J88.

References

  1. Davies, H. M. L., Du Bois, J. & Yu, J. Q. C–H functionalization in organic synthesis. Chem. Soc. Rev. 40, 1855–1856 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. Gutekunst, W. R. & Baran, P. S. C–H functionalization logic in total synthesis. Chem. Soc. Rev. 40, 1976–1991 (2011).

    Article  CAS  PubMed  Google Scholar 

  3. Yamaguchi, J., Yamaguchi, A. D. & Itami, K. C–H bond functionalization: emerging synthetic tools for natural products and pharmaceuticals. Angew. Chem. Int. Ed. 51, 8960–9009 (2012).

    Article  CAS  Google Scholar 

  4. Hartwig, J. F. Evolution of C–H bond functionalization from methane to methodology. J. Am. Chem. Soc. 138, 2–24 (2016).

    Article  CAS  PubMed  Google Scholar 

  5. Liao, K. et al. Design of catalysts for site-selective and enantioselective functionalization of non-activated primary C–H bonds. Nat. Chem. 10, 1048–1055 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Liu, Y. J. et al. Overcoming the limitation of directed C–H functionalization of heterocycles. Nature 515, 389–393 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Poulos, T. L. Heme enzyme structure and function. Chem. Rev. 114, 3919–3962 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Rudolf, J. D., Chang, C. Y., Ma, M. & Shen, B. Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function. Nat. Prod. Rep. 34, 1141–1172 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Guengerich, F. P. & Yoshimoto, F. K. Formation and cleavage of C–C bonds by enzymatic oxidation-reduction reactions. Chem. Rev. 118, 6573–6655 (2018).

    Article  CAS  PubMed  Google Scholar 

  10. Zhang, X. W. & Li, S. Y. Expansion of chemical space for natural products by uncommon P450 reactions. Nat. Prod. Rep. 34, 1061–1089 (2017).

    Article  CAS  PubMed  Google Scholar 

  11. Podust, L. M. & Sherman, D. H. Diversity of P450 enzymes in the biosynthesis of natural products. Nat. Prod. Rep. 29, 1251–1266 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Tang, M. et al. Oxidative cyclization in natural product biosynthesis. Chem. Rev. 117, 5226–5333 (2017).

    Article  CAS  PubMed  Google Scholar 

  13. Hartwig, J. F. Carbon-heteroatom bond formation catalysed by organometallic complexes. Nature 455, 314–322 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Park, Y., Kim, Y. & Chang, S. Transition metal-catalyzed C–H amination: scope, mechanism, and applications. Chem. Rev. 117, 9247–9301 (2017).

    Article  CAS  PubMed  Google Scholar 

  15. Barry, S. M. et al. Cytochrome P450-catalyzed l-tryptophan nitration in thaxtomin biosynthesis. Nat. Chem. Biol. 8, 814–816 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lin, H. C. et al. P450-mediated coupling of indole fragments to forge communesin and unnatural isomers. J. Am. Chem. Soc. 138, 4002–4005 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Irie, K. et al. The Epstein–Barr virus early antigen inducing indole alkaloids, (−)-indolactam V and its related compounds, produced by actinomycetes. Agric. Biol. Chem. 49, 1269–1274 (1984).

    Google Scholar 

  18. Chen, S. et al. Small molecule that directs differentiation of human ESCs into the pancreatic lineage. Nat. Chem. Biol. 5, 258–265 (2009).

    Article  CAS  PubMed  Google Scholar 

  19. Lim, H. J., Gallucci, J. C. & Rajanbabu, T. V. Annulzated diketopiperazines from dipeptides or Schollkopf reagents via tandem cyclization-intramolecular N-arylation. Org. Lett. 12, 2162–2165 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Nakamura, H., Yasui, K., Kanda, Y. & Baran, P. S. 11-Step total synthesis of teleocidins B-1–B-4. J. Am. Chem. Soc. 141, 1494–1497 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Edwards, D. J. & Gerwick, W. H. Lyngbyatoxin biosynthesis: sequence of biosynthetic gene cluster and identification of novel aromatic prenyltransferase. J. Am. Chem. Soc. 126, 11432–11433 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Read, J. A. & Walsh, C. T. The lyngbyatoxin biosynthetic assembly line: chain release by four-electron reduction of a dipeptidyl thioester to the corresponding alcohol. J. Am. Chem. Soc. 129, 15762–15763 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Huynh, M. U. et al. Enzymatic production of (−)-indolactam V by ltxB, a cytochrome P450 monooxynase. J. Nat. Prod. 73, 71–74 (2010).

    Article  CAS  PubMed  Google Scholar 

  24. Awakawa, T. et al. A methyltransferase initiates terpene cyclization in teleocidin B biosynthesis. J. Am. Chem. Soc. 136, 9910–9913 (2014).

    Article  CAS  PubMed  Google Scholar 

  25. Abe, I. Biosynthesis studies on teleocidins in Streptomyces. J. Antibiot. 71, 763–768 (2018).

    Article  CAS  Google Scholar 

  26. Mori, T. et al. Manipulation of prenylation reactions by structure-based engineering of bacterial indolactam prenyltransferases. Nat. Commun. 7, 10849 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Walsh, C. T. Biological matching of chemical reactivity: pairing indole nucleophilicity with electrophilic isoprenoids. ACS Chem. Biol. 9, 2718–2728 (2014).

    Article  CAS  PubMed  Google Scholar 

  28. lrie, K. et al. Synthesis of 6-substituted indolactams by microbial conversion. Tetrahedron 51, 6255–6266 (1995).

    Article  Google Scholar 

  29. Bachmann, B. O. & Ravel, J. In silico prediction of microbial secondary metabolic pathways from DNA sequence data. Methods Enzymol. 458, 181–217 (2009).

    Article  CAS  PubMed  Google Scholar 

  30. Tummino, P. J. & Copeland, R. A. Residence time of receptor–ligand complexes and its effect on biological function. Biochemistry 47, 5481–5492 (2008).

    Article  CAS  PubMed  Google Scholar 

  31. Quesenberry, M. S. & Lee, Y. C. A rapid formaldehyde assay using purpald reagent: application under periodation conditions. Anal. Biochem. 234, 50–55 (1996).

    Article  CAS  PubMed  Google Scholar 

  32. Parisi, G. et al. Substrate-induced conformational change in cytochrome P450 OleP. FASEB J. 33, 1787–1800 (2018).

    Article  PubMed  Google Scholar 

  33. Takahashi, S. et al. Structure-function analyses of cytochrome P450revI involved in reveromycin A biosynthesis and evaluation of the biological activity of its substrate, reveromycin T. J. Biol. Chem. 289, 32446–32458 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Li, Q., Chen, Y., Zhang, G. & Zhang, H. Structural analysis of SgvP involved in carbon-sulfur bond formation during griseoviridin biosynthesis. FEBS Lett. 591, 1295–1304 (2017).

    Article  CAS  PubMed  Google Scholar 

  35. Poulos, T. L. Cytochrome P450 flexibility. Proc. Natl Acad. Sci. USA 100, 13121–13122 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Skopalík, J., Anzenbacher, P. & Otyepka, M. Flexibility of human cytochromes P450: molecular dynamics reveals differences between CYPs 3A4, 2C9, and 2A6, which correlate with their substrate preferences. J. Phys. Chem. B 112, 8165–8173 (2008).

    Article  PubMed  CAS  Google Scholar 

  37. Sevrioukova, I. F. & Poulos, T. L. Structural basis for regiospecific midazolam oxidation by human cytochrome P450 3A4. Proc. Natl Acad. Sci. USA 114, 486–491 (2017).

    Article  CAS  PubMed  Google Scholar 

  38. Tietz, D. R., Podust, L. M., Sherman, D. H. & Pochapsky, T. C. Solution conformations and dynamics of substrate-bound cytochrome P450 MycG. Biochemistry 56, 2701–2714 (2017).

    Article  CAS  PubMed  Google Scholar 

  39. Davies, H. M. & Manning, J. R. Catalytic C-H functionalization by metal carbenoid and nitrenoid insertion. Nature 451, 417–424 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Singh, B. K., Polley, A. & Jana, R. Copper(II)-mediated intermolecular C(sp 2)–H amination of benzamides with electron-rich anilines. J. Org. Chem. 81, 4295–4303 (2016).

    Article  CAS  PubMed  Google Scholar 

  41. McIntosh, J. A. et al. Enantioselective intramolecular C–H amination catalyzed by engineered cytochrome P450 enzymes in vitro and in vivo. Angew. Chem. Int. Ed. 52, 9309–9312 (2013).

    Article  CAS  Google Scholar 

  42. McIntosh, J. A., Farwell, C. C. & Arnold, F. H. Expanding P450 catalytic reaction space through evolution and engineering. Curr. Opin. Chem. Biol. 19, 126–134 (2014).

    Article  CAS  PubMed  Google Scholar 

  43. Tsutsumi, H. et al. Unprecedented cyclization catalyzed by a cytochrome P450 in benzastatin biosynthesis. J. Am. Chem. Soc. 140, 6631–6639 (2018).

    Article  CAS  PubMed  Google Scholar 

  44. Onaka, H., Asamizu, S., Igarashi, Y., Yoshida, R. & Furumai, T. Cytochrome P450 homolog is responsible for C–N bond formation between aglycone and deoxysugar in the staurosporine biosynthesis of Streptomyces sp. TP-A0274. Biosci. Biotechnol. Biochem. 69, 1753–1759 (2005).

    Article  CAS  PubMed  Google Scholar 

  45. Dodani, S. C. et al. Discovery of a regioselectivity switch in nitrating P450s guided by molecular dynamics simulations and Markov models. Nat. Chem. 8, 419–425 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Alkhalaf, L. M. et al. Binding of distinct substrate conformations enables hydroxylation of remote sites in thaxtomin D by cytochrome P450 TxtC. J. Am. Chem. Soc. 141, 216–222 (2018).

    Article  PubMed  CAS  Google Scholar 

  47. Barton, D. H. R. et al. Investigations on the biosynthesis of morphine alkaloids. J. Chem. Soc. 0, 2423–2438 (1965).

    Article  CAS  Google Scholar 

  48. Johnny Hioe, J., Šakić, D., Vrček, V. & Zipse, H. The stability of nitrogen-centered radicals. Org. Biomol. Chem. 13, 157–169 (2015).

    Article  PubMed  CAS  Google Scholar 

  49. Pace, C. N., Vajdos, F., Fee, L., Grimsley, G. & Gray, T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4, 2411–2423 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Iizaka, Y., Takeda, R., Senzaki, Y., Fukumoto, A. & Anzai, Y. Cytochrome P450 enzyme RosC catalyzes a multistep oxidation reaction to form the non-active compound 20-carboxylrosamicin. FEMS Microbiol. Lett. 364, fnx110 (2017).

    Article  CAS  Google Scholar 

  51. Guengerich, F. P., Wilkey, C. J. & Phan, T. T. N. Human cytochrome P450 enzymes bind drugs and other substrates mainly through conformational-selection modes. J. Biol. Chem. 294, 10928–10941 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Heredia, V. V., Thomson, J., Nettleton, D. & Sun, S. Glucose-induced conformational changes in glucokinase mediate allosteric regulation: transient kinetic analysis. Biochemistry 45, 7553–7562 (2006).

    Article  CAS  PubMed  Google Scholar 

  53. Risky, L. et al. Oxidative demethylation of algal carbohydrates by cytochrome P450 monooxygenases. Nat. Chem. Biol. 14, 342–344 (2018).

    Article  CAS  Google Scholar 

  54. Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  PubMed  CAS  Google Scholar 

  57. Holm, L. & Laakso, L. M. Dali server update. Nucleic Acids Res. 44, W351–W355 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Tian, W., Chen, C., Lei, X., Zhao, J. & Liang, J. CASTp 3.0: computed atlas of surface topography of proteins. Nucleic Acids Res. 46, W363–W367 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Schüttelkopf, A. W. & van Aalten, D. M. PRODRG: a tool for high-throughput crystallography of protein–ligand complexes. Acta Crystallogr. D 60, 1355–1363 (2004).

    Article  PubMed  CAS  Google Scholar 

  60. Yukl, E. T., Goblirsch, B. R., Davidson, V. L. & Wilmot, C. M. Crystal structures of CO and NO adducts of MauG in complex with pre-methylamine dehydrogenase: implications for the mechanism of dioxygen activation. Biochemistry 50, 2931–2938 (2011).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank A. Arisawa (MicroBiopharm Japan) for giving us the plasmids of the P450 redox partner CamA and CamB. We thank T. Eguchi and F. Kudo (Tokyo Institute of Technology) for assistance with the stopped-flow experiments. The synchrotron radiation experiments were performed at the BL-1A, and NW12A of the Photon Factory and at beamline TPS-05A and BL15A1 at NSRRC. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI grant nos. JP16H06443, JP17H04763 and JP18K19139), Japan Science and Technology Agency (JST SICORP grant no. JPMJSC1701), Kobayashi International Scholarship Foundation and the Takeda Science Foundation.

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Author notes

  1. Miroslava Alblova

    Present address: Department of Structural Biology of Signaling Proteins, Division Biotechnology and Biomedicine Center of the Academy of Sciences and Charles University in Vestec, Institute of Physiology, The Czech Academy of Sciences, Prague, Czech Republic

  2. These authors contributed equally: Fei He, Takahiro Mori, Iori Morita.

Authors and Affiliations

  1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

    Fei He, Takahiro Mori, Iori Morita, Hitomi Nakamura, Miroslava Alblova, Shotaro Hoshino, Takayoshi Awakawa & Ikuro Abe

  2. Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Tokyo, Japan

    Takahiro Mori, Takayoshi Awakawa & Ikuro Abe

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Contributions

T.M., H.N. and I.A. designed the experiments. F.H., T.M. and M.A. performed in vitro analysis and crystallization experiments. I.M. measured pre-steady-state kinetic values. I.M. and H.N. synthesized substrate analogs. S.H. performed structure determination of enzyme reaction products. F.H., T.M., H.N., T.A. and I.A. analyzed the data. T.M. and I.A. wrote the paper.

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Correspondence to Ikuro Abe.

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Supplementary Tables 1–6 and Supplementary Figures 1–10.

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He, F., Mori, T., Morita, I. et al. Molecular basis for the P450-catalyzed C–N bond formation in indolactam biosynthesis. Nat Chem Biol 15, 1206–1213 (2019). https://doi.org/10.1038/s41589-019-0380-9

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