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:

Hydroxytryptophan biosynthesis by a family of heme-dependent enzymes in bacteria

Nature Chemical Biology volume 19, pages 1415–1422 (2023)Cite this article

Subjects

Abstract

Hydroxytryptophan serves as a chemical precursor to a variety of bioactive specialized metabolites, including the human neurotransmitter serotonin and the hormone melatonin. Although the human and animal routes to hydroxytryptophan have been known for decades, how bacteria catalyze tryptophan indole hydroxylation remains a mystery. Here we report a class of tryptophan hydroxylases that are involved in various bacterial metabolic pathways. These enzymes utilize a histidine-ligated heme cofactor and molecular oxygen or hydrogen peroxide to catalyze regioselective hydroxylation on the tryptophan indole moiety, which is mechanistically distinct from their animal counterparts from the nonheme iron enzyme family. Through genome mining, we also identify members that can hydroxylate the tryptophan indole ring at alternative positions. Our results not only reveal a conserved way to synthesize hydroxytryptophans in bacteria but also provide a valuable enzyme toolbox for biocatalysis. As proof of concept, we assemble a highly efficient pathway for melatonin in a bacterial host.

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

Fig. 1: Comparison of enzymatic routes to hydroxytryptophans in animals and bacteria.
Fig. 2: In vivo studies of starter unit biosynthesis in the luzopeptin pathway.
Fig. 3: In vitro studies of Luz16- and Luz15-catalyzed reactions.
Fig. 4: Characterization of bacterial tryptophan hydroxylases from genome mining.
Fig. 5: Biochemical characterization of selected Luz15 variants and the proposed catalytic mechanism for bacterial tryptophan hydroxylases.
Fig. 6: Microbial de novo biosynthesis of melatonin.

Similar content being viewed by others

Data availability

The data that support the findings of this study are available within the main text and its Supplementary Information files. Data are also available from the corresponding author upon request. Source data are provided with this paper.

References

  1. Alkhalaf, L. M. & Ryan, K. S. Biosynthetic manipulation of tryptophan in bacteria: pathways and mechanisms. Chem. Biol. 22, 317–328 (2015).

    CAS  PubMed  Google Scholar 

  2. Axelrod, J. & Weissbach, H. Enzymatic O-methylation of N-acetylserotonin to melatonin. Science 131, 1312–1312 (1960).

    CAS  PubMed  Google Scholar 

  3. Maeda, H. & Dudareva, N. The shikimate pathway and aromatic amino acid biosynthesis in plants. Ann. Rev. Plant Biol. 63, 73–105 (2012).

    CAS  Google Scholar 

  4. Lee, J.-H., Wood, T. K. & Lee, J. Roles of indole as an interspecies and interkingdom signaling molecule. Trends Microbiol. 23, 707–718 (2015).

    CAS  PubMed  Google Scholar 

  5. Kaur, H. et al. The antibiotic darobactin mimics a β-strand to inhibit outer membrane insertase. Nature 593, 125–129 (2021).

    CAS  PubMed  Google Scholar 

  6. Fricke, J., Blei, F. & Hoffmeister, D. Enzymatic synthesis of psilocybin. Angew. Chem. Int. Ed. Engl. 56, 12352–12355 (2017).

    CAS  PubMed  Google Scholar 

  7. Caputi, L. et al. Missing enzymes in the biosynthesis of the anticancer drug vinblastine in Madagascar periwinkle. Science 360, 1235–1239 (2018).

    CAS  PubMed  Google Scholar 

  8. Daniels, P. N. et al. A biosynthetic pathway to aromatic amines that uses glycyl-tRNA as nitrogen donor. Nat. Chem. 14, 71–77 (2022).

    CAS  PubMed  Google Scholar 

  9. Hong, B. et al. Biosynthesis of strychnine. Nature 607, 617–622 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Adak, S., Lukowski, A. L., Schäfer, R. J. B. & Moore, B. S. From tryptophan to toxin: nature’s convergent biosynthetic strategy to aetokthonotoxin. J. Am. Chem. Soc. 144, 2861–2866 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Yu, J. et al. Parallel pathways for serotonin biosynthesis and metabolism in C. elegans. Nat. Chem. Biol. https://doi.org/10.1038/s41589-022-01148-7 (2022).

  12. Fitzpatrick, P. F. Tetrahydropterin-dependent amino acid hydroxylases. Annu. Rev. Biochem. 68, 355–381 (1999).

    CAS  PubMed  Google Scholar 

  13. Walther, D. J. et al. Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 299, 76 (2003).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Winkelblech, J. et al. Tryptophan C5-, C6- and C7-prenylating enzymes displaying a preference for C-6 of the indole ring in the presence of unnatural dimethylallyl diphosphate analogues. Adv. Synth. Catal. 357, 975–986 (2015).

    CAS  Google Scholar 

  17. Tsunematsu, Y. et al. Distinct mechanisms for spiro-carbon formation reveal biosynthetic pathway crosstalk. Nat. Chem. Biol. 9, 818–825 (2013).

    CAS  PubMed  Google Scholar 

  18. Farrow, S. C. et al. Cytochrome P450 and O-methyltransferase catalyze the final steps in the biosynthesis of the anti-addictive alkaloid ibogaine from Tabernanthe iboga. J. Biol. Chem. 293, 13821–13833 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Chang, F.-Y., Ternei, M. A., Calle, P. Y. & Brady, S. F. Discovery and synthetic refactoring of tryptophan dimer gene clusters from the environment. J. Am. Chem. Soc. 135, 17906–17912 (2013).

    CAS  PubMed  Google Scholar 

  20. Muliandi, A. et al. Biosynthesis of the 4-methyloxazoline-containing nonribosomal peptides, JBIR-34 and -35, in Streptomyces sp. Sp080513GE-23. Chem. Biol. 21, 923–934 (2014).

    CAS  PubMed  Google Scholar 

  21. Hoshino, T. Violacein and related tryptophan metabolites produced by Chromobacterium violaceum: biosynthetic mechanism and pathway for construction of violacein core. Appl. Microbiol. Biotechnol. 91, 1463–1475 (2011).

    CAS  PubMed  Google Scholar 

  22. Wang, H. et al. Metabolic pathway engineering for high-level production of 5-hydroxytryptophan in Escherichia coli. Metab. Eng. 48, 279–287 (2018).

    CAS  PubMed  Google Scholar 

  23. Liu, J., Ng, T., Rui, Z., Ad, O. & Zhang, W. Unusual acetylation-dependent reaction cascade in the biosynthesis of the pyrroloindole drug physostigmine. Angew. Chem. Int. Ed. Engl. 53, 136–139 (2014).

    CAS  PubMed  Google Scholar 

  24. Smith, D. R. M. et al. An unusual flavin-dependent halogenase from the metagenome of the marine sponge Theonella swinhoei WA. ACS Chem. Biol. 12, 1281–1287 (2017).

    CAS  PubMed  Google Scholar 

  25. Shi, X. et al. Enzymatic tailoring in luzopeptin biosynthesis involves cytochrome P450-mediated carbon–nitrogen bond desaturation for hydrazone formation. Angew. Chem. Int. Ed. Engl. 60, 19821–19828 (2021).

    CAS  PubMed  Google Scholar 

  26. Hirose, Y. et al. Involvement of common intermediate 3-hydroxy-l-kynurenine in chromophore biosynthesis of quinomycin family antibiotics. J. Antibiot. 64, 117–122 (2011).

    CAS  Google Scholar 

  27. Zallot, R., Oberg, N. & Gerlt, J. A. The EFI web resource for genomic enzymology tools: leveraging protein, genome, and metagenome databases to discover novel enzymes and metabolic pathways. Biochemistry 58, 4169–4182 (2019).

    CAS  PubMed  Google Scholar 

  28. Pogorevc, D. et al. Biosynthesis and heterologous production of argyrins. ACS Synth. Biol. 8, 1121–1133 (2019).

    CAS  PubMed  Google Scholar 

  29. Kim, E. et al. Characterization of the ohmyungsamycin biosynthetic pathway and generation of derivatives with improved antituberculosis activity. Biomolecules 9, 672 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Forouhar, F. et al. Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase. Proc. Natl Acad. Sci. USA 104, 473–478 (2007).

    CAS  PubMed  Google Scholar 

  31. Basran, J. et al. The mechanism of formation of n-formylkynurenine by heme dioxygenases. J. Am. Chem. Soc. 133, 16251–16257 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Shin, I. et al. Stepwise O-atom transfer in heme-based tryptophan dioxygenase: role of substrate ammonium in epoxide ring opening. J. Am. Chem. Soc. 140, 4372–4379 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Basran, J., Booth, E. S., Lee, M., Handa, S. & Raven, E. L. Analysis of reaction intermediates in tryptophan 2,3-dioxygenase: a comparison with indoleamine 2,3-dioxygenase. Biochemistry 55, 6743–6750 (2016).

    CAS  PubMed  Google Scholar 

  34. Geng, J., Weitz, A. C., Dornevil, K., Hendrich, M. P. & Liu, A. Kinetic and spectroscopic characterization of the catalytic ternary complex of tryptophan 2,3-dioxygenase. Biochemistry 59, 2813–2822 (2020).

    CAS  PubMed  Google Scholar 

  35. Sacramento, J. J. D. & Goldberg, D. P. Oxidation of an indole substrate by porphyrin iron(III) superoxide: relevance to indoleamine and tryptophan 2,3-dioxygenases. Chem. Commun. 56, 3089–3092 (2020).

    CAS  Google Scholar 

  36. Mondal, P., Rajapakse, S. & Wijeratne, G. B. Following nature’s footprint: mimicking the high-valent heme-oxo mediated indole monooxygenation reaction landscape of heme enzymes. J. Am. Chem. Soc. 144, 3843–3854 (2022).

    CAS  PubMed  Google Scholar 

  37. Wei, Y. et al. Directed evolution of a tryptophan 2,3-dioxygenase for the diastereoselective monooxygenation of tryptophans. Angew. Chem. Int. Ed. Engl. 59, 3043–3047 (2020).

  38. Connor, K. L., Colabroy, K. L. & Gerratana, B. A heme peroxidase with a functional role as an l-tyrosine hydroxylase in the biosynthesis of anthramycin. Biochemistry 50, 8926–8936 (2011).

    CAS  PubMed  Google Scholar 

  39. Tang, M.-C., Fu, C.-Y. & Tang, G.-L. Characterization of SfmD as a heme peroxidase that catalyzes the regioselective hydroxylation of 3-methyltyrosine to 3-hydroxy-5-methyltyrosine in saframycin A biosynthesis. J. Biol. Chem. 287, 5112–5121 (2012).

    CAS  PubMed  Google Scholar 

  40. Shin, I., Wang, Y. & Liu, A. A new regime of heme-dependent aromatic oxygenase superfamily. Proc. Natl Acad. Sci. USA 118, e2106561118 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Shin, I. et al. A novel catalytic heme cofactor in SfmD with a single thioether bond and a bis-His ligand set revealed by a de novo crystal structural and spectroscopic study. Chem. Sci. 12, 3984–3998 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Sambrook, J. Molecular Cloning: A Laboratory Manual (CSHL Press, 2001).

  43. Kieser, T. Practical Streptomyces Genetics (John Innes Foundation, 2000).

  44. Du, Y.-L., Dalisay, D. S., Andersen, R. J. & Ryan, K. S. N-Carbamoylation of 2,4-diaminobutyrate reroutes the outcome in padanamide biosynthesis. Chem. Biol. 20, 1002–1011 (2013).

    CAS  PubMed  Google Scholar 

  45. Barr, I. & Guo, F. Pyridine hemochromagen assay for determining the concentration of heme in purified protein solutions. Bio Protoc. 5, e1594 (2015).

    PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by National Key R&D Program of China (2018YFA0903200) and the National Natural Science Foundation of China (32122005) to Y.-L.D.

Author information

Author notes

  1. These authors contributed equally: Xinjie Shi, Guiyun Zhao.

Authors and Affiliations

  1. The Fourth Affiliated Hospital and Department of Microbiology, School of Medicine, Zhejiang University, Hangzhou, China

    Xinjie Shi, Guiyun Zhao, Zhijie Zhao, Wei Li & Yi-Ling Du

  2. Department of Pharmacy, The Fourth Affiliated Hospital, School of Medicine, Zhejiang University, Yiwu, China

    Guiyun Zhao, Miaolian Wu & Yi-Ling Du

  3. Polytechnic Institute, Zhejiang University, Hangzhou, China

    Hu Li

  4. Jinan Microecological Biomedicine Shandong Laboratory, Jinan, China

    Yi-Ling Du

  5. Zhejiang Provincial Key Laboratory for Microbial Biochemistry and Metabolic Engineering, Hangzhou, China

    Yi-Ling Du

Authors
  1. Xinjie Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Guiyun Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hu Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhijie Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Miaolian Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yi-Ling Du
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X.S. and G.Z. carried out bioinformatic, genetic and biochemical work and performed metabolic analysis, compound isolation and structure elucidation. H.L., Z.Z. and W.L. carried out metabolic engineering, metabolic analysis and flask fermentation. X.S., M.W. and Y.-L.D. designed the study, analyzed results and wrote the paper.

Corresponding author

Correspondence to Yi-Ling Du.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Chemical Biology thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

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

Extended data

Extended Data Fig. 1 In vitro biochemical assays and kinetic study of Luz15 with Trp in the presence of O2 and ascorbate.

a, Time-course studies of the in vitro reaction of Luz15 by HPLC. The conversion rate at 24 h is 12%. b, Kinetic study of Luz15 with Trp in the presence of O2 and ascorbate (25 mM). The kinetic parameters are Km = 3.1 ± 4 mM and kcat = 2.2 ± 0.1 h−1. Assays were run in triplicate and kinetic values throughout this paper are reported as mean ± s.d. of three replicates. Note: the kinetic study of Luz15 with H2O2 was not performed due to the rapid inactivation of Luz15 (the formation of the product plateaued after the first minute of turnover). However, these values were determined for other more stable Luz15 homologues (see the below Extended Data Figs).

Source data

Extended Data Fig. 2 In vitro biochemical assays and kinetic studies of PsmG.

a, Time-course studies of the in vitro reaction of PsmG with Trp in the presence of O2 and ascorbate by HPLC. The conversion rate at 24 h is 5%. b, Kinetic study of PsmG with Trp in the presence of H2O2 (3 mM). The kinetic parameters are Km = 406 ± 77 μM and kcat = 10.8 ± 0.5 min−1. c, Kinetic study of PsmG with Trp in the presence of O2 and ascorbate (25 mM). The kinetic parameters are Km = 1.7 ± 0.4 mM and kcat = 2.1 ± 0.2 h−1. Assays were run in triplicate, and data are reported as mean ± s.d. of three replicates.

Source data

Extended Data Fig. 3 In vitro biochemical assays and kinetic studies of KerI.

a, Time-course studies of the in vitro reaction of KerI with Trp in the presence of O2 and ascorbate by HPLC. The conversion rate at 24 h is 22%. b, Kinetic study of KerI with Trp in the presence of H2O2 (3 mM). The kinetic parameters are Km = 2.6 ± 0.3 mM and kcat = 87 ± 5 min−1. c, Kinetic study of KerI with Trp in the presence of O2 and ascorbate (25 mM). The kinetic parameters are Km = 448 ± 57 μM and kcat = 2.4 ± 0.1 h−1. Assays were run in triplicate and data are reported as mean ± s.d. of three replicates. It is worth mentioning that, among the Trp 5-hydroxylases we identified, KerI is the most stable protein under in vitro conditions. KerI does not precipitate at the concentrations of H2O2 we used (up to 4 mM) and has the highest conversion rate.

Source data

Extended Data Fig. 4 In vivo production and identification of 4-HTP and 6-HTP.

a, Metabolic profiling of the E. coli strain expressing arg5 and SaT6H (a Trp 6-hydoxylase, see later in the main text) by HPLC analysis (detection wavelength: 254 nm). Assays were run in triplicate and representative results are shown. b, Mass value for the strain-specific peak from E. coli (ΔtnaA)/pET22b-arg5. c, Mass value for the strain-specific peak from E. coli (ΔtnaA)/pET22b-SaT6H. d, Structure elucidation of isolated 4-HTP and 6-HTP. Key COSY and HMBC correlations are displayed.

Extended Data Fig. 5 In vitro biochemical assays of OhmK and Arg5 in the presence of O2 and ascorbate.

a, Time-course studies of the in vitro reaction of OhmK with Trp in the presence of O2 and ascorbate by HPLC. b, Time-course studies of the in vitro reaction of Arg5 with Trp in the presence of O2 and ascorbate by HPLC. c, The reaction product 4-HTP undergoes further side reactions to form blue compounds in the reaction with H2O2. This blueing reaction likely occurs through a similar oxidative oligomerization mechanism to that of psilocin (4-hydroxy-N,N-dimethyltryptamine) from mushrooms (please also see Supplementary Fig. 24). Note: the kinetic parameters of the Trp 4-hydroxylases were not determined due to the above oligomerization side reaction with H2O2, and the low conversion rates with O2 and ascorbate (approximately one turnover). Assays were run in triplicate, and representative results are shown.

Extended Data Fig. 6 In vitro biochemical assays and kinetic studies of SaT6H.

a, Time-course studies of the in vitro reaction of SaT6H with Trp in the presence of O2 and ascorbate by HPLC. The conversion rate at 30 h is 86%. b, Kinetic study of SaT6H with Trp in the presence of H2O2 (3 mM). The kinetic parameters are Km = 4.3 ± 0.9 mM and kcat = 113 ± 12 min−1.c, Kinetic study of SaT6H with Trp in the presence of O2 and ascorbate (25 mM). The kinetic parameters are Km = 1.1 ± 0.2 mM and kcat = 23 ± 2 h−1. Assays were run in triplicate, and data are reported as mean ± s.d. of three replicates.

Source data

Extended Data Fig. 7 In vitro biochemical assays and kinetic studies of AxT6H.

a, Time-course studies of the in vitro reaction of AxT6H with Trp in the presence of O2 and ascorbate by HPLC. The conversion rate at 30 h is 37%. b, Kinetic study of AxT6H with Trp in the presence of H2O2 (3 mM). The kinetic parameters are Km = 1.3 ± 0.4 mM and kcat = 31 ± 5 min−1.c, Kinetic study of AxT6H with Trp in the presence of O2 and ascorbate (25 mM). The kinetic parameters are Km = 994 ± 99 μM and kcat = 2.5 ± 0.1 h−1. Assays were run in triplicate, and data are reported as mean ± s.d. of three replicates.

Source data

Extended Data Fig. 8 Summary of substrate specificity studies of bacterial Trp hydroxylases T5H (KerI), T4H (OhmK), and T6H (SaT6H).

The conversion rates for each reaction are displayed in red. N.D. indicates ‘not detected’ (also see Supplementary Figs. 25-31).

Supplementary information

Supplementary Information

Supplementary Figs. 1–61 and Tables 1–7.

Reporting Summary

Supplementary Data 1

DNA sequences for synthetic genes.

Supplementary Data 2

Statistical source data for Supplementary Fig. 35.

Source data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shi, X., Zhao, G., Li, H. et al. Hydroxytryptophan biosynthesis by a family of heme-dependent enzymes in bacteria. Nat Chem Biol 19, 1415–1422 (2023). https://doi.org/10.1038/s41589-023-01416-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-023-01416-0

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