Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Molecular insights into the enzyme promiscuity of an aromatic prenyltransferase

Nature Chemical Biology volume 13, pages 226–234 (2017)Cite this article

Subjects

Abstract

Aromatic prenyltransferases (aPTases) transfer prenyl moieties from isoprenoid donors to various aromatic acceptors, some of which have the rare property of extreme enzymatic promiscuity toward both a variety of prenyl donors and a large diversity of acceptors. In this study, we discovered a new aPTase, AtaPT, from Aspergillus terreus that exhibits unprecedented promiscuity toward diverse aromatic acceptors and prenyl donors and also yields products with a range of prenylation patterns. Systematic crystallographic studies revealed various discrete conformations for ligand binding with donor-dependent acceptor specificity and multiple binding sites within a spacious hydrophobic substrate-binding pocket. Further structure-guided mutagenesis of active sites at the substrate-binding pocket is responsible for altering the specificity and promiscuity toward substrates and the diversity of product prenylations. Our study reveals the molecular mechanism underlying the promiscuity of AtaPT and suggests an efficient protein engineering strategy to generate new prenylated derivatives in drug discovery applications.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structures of the aromatic compounds assayed as prenyl acceptors.
Figure 2: Prenylation of aromatics by AtaPT.
Figure 3: Crystal structure of AtaPT and its catalytic chamber.
Figure 4: Donor-dependent acceptor binding by AtaPT.
Figure 5: Conformational dynamics of the tyrosine shield and various acceptor binding sites of AtaPT.
Figure 6: Structure-guided mutagenesis of AtaPT to alter its substrate promiscuity.

Similar content being viewed by others

Accession codes

Primary accessions

GenBank/EMBL/DDBJ

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Brandt, W. et al. Molecular and structural basis of metabolic diversity mediated by prenyldiphosphate converting enzymes. Phytochemistry 70, 1758–1775 (2009).

    CAS  PubMed  Google Scholar 

  2. Heide, L. Prenyl transfer to aromatic substrates: genetics and enzymology. Curr. Opin. Chem. Biol. 13, 171–179 (2009).

    CAS  PubMed  Google Scholar 

  3. Li, S.M. Prenylated indole derivatives from fungi: structure diversity, biological activities, biosynthesis and chemoenzymatic synthesis. Nat. Prod. Rep. 27, 57–78 (2010).

    PubMed  Google Scholar 

  4. Yazaki, K., Sasaki, K. & Tsurumaru, Y. Prenylation of aromatic compounds, a key diversification of plant secondary metabolites. Phytochemistry 70, 1739–1745 (2009).

    CAS  PubMed  Google Scholar 

  5. Botta, B., Vitali, A., Menendez, P., Misiti, D. & Delle Monache, G. Prenylated flavonoids: pharmacology and biotechnology. Curr. Med. Chem. 12, 717–739 (2005).

    PubMed  Google Scholar 

  6. Li, S.M. Applications of dimethylallyltryptophan synthases and other indole prenyltransferases for structural modification of natural products. Appl. Microbiol. Biotechnol. 84, 631–639 (2009).

    CAS  PubMed  Google Scholar 

  7. Kuzuyama, T., Noel, J.P. & Richard, S.B. Structural basis for the promiscuous biosynthetic prenylation of aromatic natural products. Nature 435, 983–987 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Saleh, O., Haagen, Y., Seeger, K. & Heide, L. Prenyl transfer to aromatic substrates in the biosynthesis of aminocoumarins, meroterpenoids and phenazines: the ABBA prenyltransferase family. Phytochemistry 70, 1728–1738 (2009).

    CAS  PubMed  Google Scholar 

  9. Chooi, Y.H. et al. Discovery and characterization of a group of fungal polycyclic polyketide prenyltransferases. J. Am. Chem. Soc. 134, 9428–9437 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Yu, X. & Li, S.M. Prenyltransferases of the dimethylallyltryptophan synthase superfamily. Methods Enzymol. 516, 259–278 (2012).

    CAS  PubMed  Google Scholar 

  11. Rudolf, J.D., Wang, H. & Poulter, C.D. Multisite prenylation of 4-substituted tryptophans by dimethylallyltryptophan synthase. J. Am. Chem. Soc. 135, 1895–1902 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Ding, Y. et al. Genome-based characterization of two prenylation steps in the assembly of the stephacidin and notoamide anticancer agents in a marine-derived Aspergillus sp. J. Am. Chem. Soc. 132, 12733–12740 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Cheng, W. & Li, W. Structural insights into ubiquinone biosynthesis in membranes. Science 343, 878–881 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Kumano, T., Richard, S.B., Noel, J.P., Nishiyama, M. & Kuzuyama, T. Chemoenzymatic syntheses of prenylated aromatic small molecules using Streptomyces prenyltransferases with relaxed substrate specificities. Bioorg. Med. Chem. 16, 8117–8126 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Zhou, K., Yu, X., Xie, X. & Li, S.M. Complementary flavonoid prenylations by fungal indole prenyltransferases. J. Nat. Prod. 78, 2229–2235 (2015).

    CAS  PubMed  Google Scholar 

  16. Yin, S., Yu, X., Wang, Q., Liu, X.Q. & Li, S.M. Identification of a brevianamide F reverse prenyltransferase BrePT from Aspergillus versicolor with a broad substrate specificity towards tryptophan-containing cyclic dipeptides. Appl. Microbiol. Biotechnol. 97, 1649–1660 (2013).

    CAS  PubMed  Google Scholar 

  17. Kremer, A., Westrich, L. & Li, S.M. A 7-dimethylallyltryptophan synthase from Aspergillus fumigatus: overproduction, purification and biochemical characterization. Microbiology 153, 3409–3416 (2007).

    CAS  PubMed  Google Scholar 

  18. Schuller, J.M. et al. Structure and catalytic mechanism of a cyclic dipeptide prenyltransferase with broad substrate promiscuity. J. Mol. Biol. 422, 87–99 (2012).

    CAS  PubMed  Google Scholar 

  19. Pockrandt, D., Sack, C., Kosiol, T. & Li, S.M. A promiscuous prenyltransferase from Aspergillus oryzae catalyses C-prenylations of hydroxynaphthalenes in the presence of different prenyl donors. Appl. Microbiol. Biotechnol. 98, 4987–4994 (2014).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Shen, Y. et al. Butyrolactone and cycloheptanetrione from mangrove-associated fungus Aspergillus terreus. Chem. Pharm. Bull. (Tokyo) 60, 1437–1441 (2012).

    CAS  Google Scholar 

  22. Gao, B., Chen, R., Liu, X., Dai, J. & Sun, F. Expression, purification, crystallization and crystallographic study of the Aspergillus terreus aromatic prenyltransferase AtaPT. Acta Crystallogr. F Struct. Biol. Commun. 71, 889–894 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Metzger, U. et al. The structure of dimethylallyl tryptophan synthase reveals a common architecture of aromatic prenyltransferases in fungi and bacteria. Proc. Natl. Acad. Sci. USA 106, 14309–14314 (2009).

    CAS  PubMed  Google Scholar 

  24. Nitta, K., Fujita, N., Yoshimura, T., Arai, K. & Yamamoto, Y. Metabolic products of Aspergillus terreus. IX. Biosynthesis of butyrolactone derivatives isolated from strains IFO 8835 and 4100. Chem. Pharm. Bull. 31, 1528–1533 (1983).

    CAS  Google Scholar 

  25. Guo, C.J. et al. Spatial regulation of a common precursor from two distinct genes generates metabolite diversity. Chem. Sci. 6, 5913–5921 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Markovits, J. et al. Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerase II. Cancer Res. 49, 5111–5117 (1989).

    CAS  PubMed  Google Scholar 

  27. Pavese, J.M., Krishna, S.N. & Bergan, R.C. Genistein inhibits human prostate cancer cell detachment, invasion, and metastasis. Am. J. Clin. Nutr. 100 (Suppl. 1), 431S–436S (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D. & Mitchell, J.B. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 47, 936–942 (1987).

    CAS  PubMed  Google Scholar 

  29. Yu, X. et al. Catalytic mechanism of stereospecific formation of cis-configured prenylated pyrroloindoline diketopiperazines by indole prenyltransferases. Chem. Biol. 20, 1492–1501 (2013).

    CAS  PubMed  Google Scholar 

  30. Jost, M. et al. Structure-function analysis of an enzymatic prenyl transfer reaction identifies a reaction chamber with modifiable specificity. J. Am. Chem. Soc. 132, 17849–17858 (2010).

    CAS  PubMed  Google Scholar 

  31. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    CAS  PubMed  Google Scholar 

  32. Khersonsky, O. & Tawfik, D.S. Enzyme promiscuity: a mechanistic and evolutionary perspective. Annu. Rev. Biochem. 79, 471–505 (2010).

    CAS  PubMed  Google Scholar 

  33. Chen, D. et al. Probing the catalytic promiscuity of a regio- and stereospecific C-glycosyltransferase from Mangifera indica. Angew. Chem. Int. Ed. Engl. 54, 12678–12682 (2015).

    CAS  PubMed  Google Scholar 

  34. Cuetos, A. et al. Catalytic promiscuity of transaminases: preparation of enantioenriched β-fluoroamines by formal tandem hydrodefluorination/deamination. Angew. Chem. Int. Ed. 55, 3144–3147 (2016).

    CAS  Google Scholar 

  35. Ekroos, M. & Sjögren, T. Structural basis for ligand promiscuity in cytochrome P450 3A4. Proc. Natl. Acad. Sci. USA 103, 13682–13687 (2006).

    CAS  PubMed  Google Scholar 

  36. Schmid, A. et al. Industrial biocatalysis today and tomorrow. Nature 409, 258–268 (2001).

    CAS  PubMed  Google Scholar 

  37. Ho, B.K. & Gruswitz, F. HOLLOW: generating accurate representations of channel and interior surfaces in molecular structures. BMC Struct. Biol. 8, 49 (2008).

    PubMed  PubMed Central  Google Scholar 

  38. Woodside, A.B., Huang, Z. & Poulter, C.D. Trisammonium geranyl diphosphate. [diphosphoric acid, mono(3,7-dimethyl-2,6-octadienyl) ester (E)-, trisammonium salt]. Org. Synth. 66, 211 (1988).

    CAS  Google Scholar 

  39. Braña, M.F. et al. Synthesis and biological evaluation of analogues of butyrolactone I and molecular model of its interaction with CDK2. Org. Biomol. Chem. 2, 1864–1871 (2004).

    PubMed  Google Scholar 

  40. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Pettersen, E.F. et al. UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    CAS  Google Scholar 

  43. Phillips, J.C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. MacKerell, A.D. et al. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 102, 3586–3616 (1998).

    CAS  PubMed  Google Scholar 

  45. MacKerell, A.D. Jr., Feig, M. & Brooks, C.L., III. Improved treatment of the protein backbone in empirical force fields. J. Am. Chem. Soc. 126, 698–699 (2004).

    CAS  PubMed  Google Scholar 

  46. Best, R.B. et al. Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone ϕ, ψ and side-chain χ(1) and χ(2) dihedral angles. J. Chem. Theory Comput. 8, 3257–3273 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Yu, W., He, X., Vanommeslaeghe, K. & MacKerell, A.D. Jr. Extension of the CHARMM General Force Field to sulfonyl-containing compounds and its utility in biomolecular simulations. J. Comput. Chem. 33, 2451–2468 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38, 27–28 (1996).

    CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Huang and J. He for their on-site assistance with the crystallographic data collection at the SSRF beamline BL17U. This work was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB08030202) to F.S., the National Basic Research Program (973 Program) of Ministry of Science and Technology of China (2014CB910700) to F.S. and (2014CB910202) to J.L., the National Natural Science Foundation of China (81302667) to R.C. and the Deutsche Forschungsgemeinschaft (Li844/4-1) to S.-M.L. The computational resources were provided by the National Supercomputing Center in Tianjin and the HPC-Service Station at the Center for Biological Imaging of the Institute of Biophysics of the Chinese Academy of Sciences.

Author information

Author notes

  1. Xiao Liu

    Present address: Present address: Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China.,

  2. Ridao Chen, Bingquan Gao and Xiao Liu: These authors contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Ridao Chen, Xiao Liu, Feiying Ruan, Keping Feng & Jungui Dai

  2. National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Bingquan Gao & Fei Sun

  3. University of Chinese Academy of Sciences, Beijing, China

    Bingquan Gao & Fei Sun

  4. Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Yong Zhang & Jizhong Lou

  5. Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Marburg, Germany

    Carsten Wunsch & Shu-Ming Li

Authors
  1. Ridao Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bingquan Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiao Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Feiying Ruan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jizhong Lou
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Keping Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Carsten Wunsch
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Shu-Ming Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jungui Dai
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Fei Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.D., S.-M.L. and F.S. supervised the project. R.C., X.L., F.R., C.W. and K.F. performed the enzymatic and chemical experiments. B.G. performed the crystallographic and ITC experiments. Y.Z. and J.L. performed the molecular dynamics simulations. R.C., X.L., B.G., J.D. and F.S. analyzed the data and wrote the manuscript.

Corresponding authors

Correspondence to Jungui Dai or Fei Sun.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–6 and Supplementary Figures 1–17. (PDF 2879 kb)

Supplementary Note

The spectroscopic data of identified catalytic products by AtaPT and the corresponding spectra. (PDF 10265 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, R., Gao, B., Liu, X. et al. Molecular insights into the enzyme promiscuity of an aromatic prenyltransferase. Nat Chem Biol 13, 226–234 (2017). https://doi.org/10.1038/nchembio.2263

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2263

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing