A marine viral halogenase that iodinates diverse substrates

Abstract

Oceanic cyanobacteria are the most abundant oxygen-generating phototrophs on our planet and are therefore important to life. These organisms are infected by viruses called cyanophages, which have recently shown to encode metabolic genes that modulate host photosynthesis, phosphorus cycling and nucleotide metabolism. Herein we report the characterization of a wild-type flavin-dependent viral halogenase (VirX1) from a cyanophage. Notably, halogenases have been previously associated with secondary metabolism, tailoring natural products. Exploration of this viral halogenase reveals it capable of regioselective halogenation of a diverse range of substrates with a preference for forming aryl iodide species; this has potential implications for the metabolism of the infected host. Until recently, a flavin-dependent halogenase that is capable of iodination in vitro had not been reported. VirX1 is interesting from a biocatalytic perspective as it shows strikingly broad substrate flexibility and a clear preference for iodination, as illustrated by kinetic analysis. These factors together render it an attractive tool for synthesis.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: A protein-based branching analysis of VirX1 against known FDHs and other non-FDH flavoenzymes that reveals the functional relationship, known substrate utilization and branching order between protein clusters.
Fig. 2: The diverse substrate scope of VirX1.
Fig. 3: Crystal structure and homotrimer assembly of VirX1.
Fig. 4: Structural comparison of VirX1 to the flavin-dependent halogenases PrnA (2AQJ) and BrvH (6FRL).

Data availability

The data that support the findings of this study are available in this Article and its Supplementary Information, or are available from the corresponding author on reasonable request. The structural factors and coordinates of the VirX1 have been deposited in the PDB (PDB ID: 6QGM).

References

  1. 1.

    Agarwal, V. et al. Enzymatic halogenation and dehalogenation reactions: pervasive and mechanistically diverse. Chem. Rev. 117, 5619–5674 (2017).

    CAS  Article  Google Scholar 

  2. 2.

    Gkotsi, D. S., Dhaliwal, J., McLachlan, M. M., Mulholand, K. R. & Goss, R. J. M. Halogenases: powerful tools for biocatalysis (mechanisms applications and scope). Curr. Opin. Chem. Biol. 43, 119–126 (2018).

    CAS  Article  Google Scholar 

  3. 3.

    Weichold, V., Milbredt, D. & van Pée, K.-H. Specific enzymatic halogenation-from the discovery of halogenated enzymes to their applications in vitro and in vivo. Angew. Chem. Int. Ed. 55, 6374–6389 (2016).

    CAS  Article  Google Scholar 

  4. 4.

    Dong, C. et al. Tryptophan 7-halogenase (PrnA) structure suggests a mechanism for regioselective chlorination. Science 309, 2216–2219 (2005).

    CAS  Article  Google Scholar 

  5. 5.

    Keller, S. et al. Purification and partial characterization of tryptophan 7-halogenase (PrnA) from Pseudomonas fluorescence. Angew. Chem. Int. Ed. 39, 2300–2302 (2000).

    CAS  Article  Google Scholar 

  6. 6.

    Dong, C. J. et al. Crystal structure and mechanism of a bacterial fluorinating enzyme. Nature 427, 561–565 (2004).

    CAS  Article  Google Scholar 

  7. 7.

    Mori, S., Pang, A. H., Chandrika, N. T., Garneau-Tsodikova, S. & Tsodikov, O. V. Unusual substrate and halide versatility of phenolic halogenase PltM. Nat. Commun. 10, 1255 (2019).

    Article  Google Scholar 

  8. 8.

    Zeng, J. & Zhan, J. A novel fungal flavin-dependent halogenase for natural product biosynthesis. ChemBioChem. 11, 2119–2123 (2010).

    CAS  Article  Google Scholar 

  9. 9.

    Neumann, C. S., Walsh, C. T. & Kay, R. R. A flavin-dependent halogenase catalyzes the chlorination step in the biosynthesis of Dictyostelium differentiation-inducing factor 1. Proc. Natl Acad. Sci. USA 107, 5798–5803 (2010).

    CAS  Article  Google Scholar 

  10. 10.

    Lang, A. et al. Changing the regioselectivity of the tryptophan 7-halogenase PrnA by site directed mutagenesis. Angew. Chem. Int. Ed. 50, 2951–2951 (2011).

    CAS  Article  Google Scholar 

  11. 11.

    Glenn, W. S., Nims, E. & O’Connor, S. E. Reengineering a tryptophan halogenase to preferentially chlorinate a direct alkaloid precursor. J. Am. Chem. Soc. 133, 19348–19349 (2011).

    Article  Google Scholar 

  12. 12.

    Payne, J. T., Poor, C. B. & Lewis, J. C. Directed evolution of RebH for site selective halogenation of large biologically active molecules. Angew. Chem. Int. Ed. 54, 4226–4230 (2015).

    CAS  Article  Google Scholar 

  13. 13.

    Menon, B. R. K. et al. RadH: a versatile halogenase for integration into synthetic pathways. Angew. Chem. Int. Ed. 56, 11841–11845 (2017).

    CAS  Article  Google Scholar 

  14. 14.

    Schnepel, C., Mignes, H., Frese, M. & Sewald, N. A high-throughput fluorescence assay to determine the activity of tryptophan halogenases. Angew. Chem. Int. Ed. 55, 14159–14163 (2017).

    Article  Google Scholar 

  15. 15.

    Goss, R. J. M. & Gkotsi, D. S. Discovery and utilisation of wildly different halogenases, powerful new tools for medicinal chemistry. UK patent GB1803491.8 (2018).

  16. 16.

    Agarwal, V. et al. Biosynthesis of polybrominated aromatic organic compounds by marine bacteria. Nat. Chem. Bio. 10, 640–647 (2014).

    CAS  Article  Google Scholar 

  17. 17.

    Sullivan, M. B., Waterbury, J. B. & Chisholm, S. W. Cyanophage infecting the oceanic cyanobacterium Prochlorococcus. Nature 424, 1047–1051 (2003).

    CAS  Article  Google Scholar 

  18. 18.

    Rost, B. Twilight zone of protein sequence alignments. Protein Engineering 12, 85–94 (1999).

    CAS  Article  Google Scholar 

  19. 19.

    Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).

    CAS  Article  Google Scholar 

  20. 20.

    James, M. J., Cuthbertson, J. D., O’Brien, P., Taylor, R. J. K. & Unsworth, W. P. Silver(i) or copper(ii)-mediated dearomatisation of aromatic ynones: direct access to spirocyclic scaffolds. Angew. Chem. Int. Ed. 54, 7640–7643 (2015).

    CAS  Article  Google Scholar 

  21. 21.

    Chambers, S. J. et al. Heteroaromatic acids and imines to azaspirocycles: stereoselective synthesis and 3D shape analysis. Chem. Eur. J. 22, 6496–6500 (2016).

    CAS  Article  Google Scholar 

  22. 22.

    Yeh, E., Blasiak, L. C., Koglin, A., Drennan, C. L. & Walsh, C. T. Chlorination by a long-lived intermediate in the mechanism of flavin-dependent halogenases. Biochem. 46, 1284–1292 (2007).

    CAS  Article  Google Scholar 

  23. 23.

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

    CAS  Article  Google Scholar 

  24. 24.

    Krissinel, E. Stock-based detection of protein oligomeric states in jsPISA. Nucl. Acids Res. 43, W314–W319 (2015).

    CAS  Article  Google Scholar 

  25. 25.

    Neubauer, P. R. et al. A flavin-dependent halogenase from metagenomic analysis prefers bromination over chlorination. PLoS ONE 13, e0196797 (2018).

    Article  Google Scholar 

  26. 26.

    Sharma, S. V. et al. Living GenoChemetics: hyphenating synthetic biology and synthetic chemistry in vivo. Nat. Commun. 8, 229 (2017).

    Article  Google Scholar 

  27. 27.

    Breitbart, M., Bonnain, C., Malki, K. & Sawaya, N. A. Phage puppet masters of the marine microbial relm. Nat. Microbiol. 3, 754–766 (2018).

    CAS  Article  Google Scholar 

  28. 28.

    Amachi, S. Microbial contribution to global iodine cycling: volatilization, accumulation, reduction, oxidation and sorption of iodine. Microbes. Environ. 23, 269–276 (2008).

    Article  Google Scholar 

  29. 29.

    Crockford, S. J. Evolutionary roots of iodine and thyroid hormones in cell-cell signalling. Integr. Com. Biol. 49, 155–166 (2009).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007–2013/ERC grant agreement no. 614779 GenoChemetics to R.J.M.G.), Syngenta and Wellcome ISSF (grant no. 204821/Z/16/Z to D.S.G.) for generous financial support. We thank G. Harris and M. Weckener (Harwell) for size-excluion chromatography multiangle light scattering and analytical ultracentrifugation analysis. We thank all of our colleagues, in particular, T. Smith and co-workers in the School of Chemistry and the Biomedical Sciences Research Complex at the University of St Andrews for all of the help that they have afforded us in the aftermath of the BMS fire. We thank I. M. Wilson for assistance with graphics.

Author information

Affiliations

Authors

Contributions

D.S.G. and R.J.M.G. conceived and designed the experiments, and the full programme was carried out under the guidance and direction of R.J.M.G. D.S.G. identified VirX1 bioinformatically, established its protein production and purification, determined the iodinase activity and carried out its biochemical investigation and substrate screening. D.S.G. and H.L. carried out the structural analysis of the enzyme under the guidance of J.H.N. D.S.G. and S.V.S. explored the differential reactivity of the substrates with hypoiodous acid, characterized the products of the iodinase and synthesized standards for comparison to products. W.P.U. and R.J.K.T. synthesized a series of spiroindolic compounds and their derivatives, which were utilized as substrates by the enzyme. M.M.W.M. and S.S. contributed to the selection of compounds for the assaying of the iodinase. H.L., J.A.C. and J.H.N. carried out substrate docking to the iodinase. D.S.G. and J.D. assayed PrnA. D.S.G., H.L., J.A.C., J.D. and Y.W. assisted with cloning and protein production. R.J.M.G., S.V.S., D.S.G., H.L. and J.A.C. wrote the paper with contributions from all authors.

Corresponding author

Correspondence to Rebecca J. M. Goss.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Experimental methods, tables and figures.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Gkotsi, D.S., Ludewig, H., Sharma, S.V. et al. A marine viral halogenase that iodinates diverse substrates. Nat. Chem. 11, 1091–1097 (2019). https://doi.org/10.1038/s41557-019-0349-z

Download citation

Further reading

Search

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