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.

  • Letter
  • Published:

Structural basis for cofactor-independent dioxygenation in vancomycin biosynthesis

Nature volume 447, pages 342–345 (2007)Cite this article

Abstract

Enzyme-catalysed oxidations are some of the most common transformations in primary and secondary metabolism. The vancomycin biosynthetic enzyme DpgC belongs to a small class of oxygenation enzymes that are not dependent on an accessory cofactor or metal ion1. The detailed mechanism of cofactor-independent oxygenases has not been established. Here we report the first structure of an enzyme of this oxygenase class in complex with a bound substrate mimic. The use of a designed, synthetic substrate analogue allows unique insights into the chemistry of oxygen activation. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is present adjacent to the site of oxidation on the substrate. Molecular oxygen is bound in a small hydrophobic pocket and the substrate provides the reducing power to activate oxygen for downstream chemical steps. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics2. Furthermore, mechanistic parallels exist between DpgC and cofactor-dependent flavoenzymes3, providing information regarding the general mechanism of enzymatic oxygen activation.

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: The dioxygenase DpgC.
Figure 4: Proposed catalytic mechanism.
Figure 2: The X-ray structure of DpgC.
Figure 3: Substrate recognition and active site of DpgC.

Similar content being viewed by others

References

  1. Tseng, C. C., Vaillancourt, F. H., Bruner, S. D. & Walsh, C. T. DpgC is a metal- and cofactor-free 3,5-dihydroxyphenylacetyl-CoA 1,2-dioxygenase in the vancomycin biosynthetic pathway. Chem. Biol. 11, 1195–1203 (2004)

    Article  CAS  Google Scholar 

  2. Walsh, C. T. Polyketide and nonribosomal peptide antibiotics: modularity and versatility. Science 303, 1805–1810 (2004)

    Article  ADS  CAS  Google Scholar 

  3. Eswaramoorthy, S., Bonanno, J. B., Burley, S. K. & Swaminathan, S. Mechanism of action of a flavin-containing monooxygenase. Proc. Natl Acad. Sci. USA 103, 9832–9837 (2006)

    Article  ADS  CAS  Google Scholar 

  4. Silverman, R. B. (ed.) The Organic Chemistry of Enzyme-Catalyzed Reactions. Revised edn. (Academic Press, San Diego, 2002)

    Google Scholar 

  5. Cane, D. E., Walsh, C. T. & Khosla, C. Harnessing the biosynthetic code: combinations, permutations, and mutations. Science 282, 63–68 (1998)

    Article  CAS  Google Scholar 

  6. Hubbard, B. K. & Walsh, C. T. Vancomycin assembly: nature's way. Angew. Chem. Int. Edn Engl. 42, 730–765 (2003)

    Article  CAS  Google Scholar 

  7. Chen, H., Tseng, C. C., Hubbard, B. K. & Walsh, C. T. Glycopeptide antibiotic biosynthesis: enzymatic assembly of the dedicated amino acid monomer (S)-3,5-dihydroxyphenylglycine. Proc. Natl Acad. Sci. USA 98, 14901–14906 (2001)

    Article  ADS  CAS  Google Scholar 

  8. Agnihotri, G. & Liu, H. W. Enoyl-CoA hydratase. reaction, mechanism, and inhibition. Bioorg. Med. Chem. 11, 9–20 (2003)

    Article  CAS  Google Scholar 

  9. Holden, H. M., Benning, M. M., Haller, T. & Gerlt, J. A. The crotonase superfamily: divergently related enzymes that catalyze different reactions involving acyl coenzyme A thioesters. Acc. Chem. Res. 34, 145–157 (2001)

    Article  CAS  Google Scholar 

  10. Beaman, T. W. et al. Acyl group specificity at the active site of tetrahydridipicolinate N-succinyltransferase. Protein Sci. 11, 974–979 (2002)

    Article  CAS  Google Scholar 

  11. Xiang, H., Luo, L., Taylor, K. L. & Dunaway-Mariano, D. Interchange of catalytic activity within the 2-enoyl-coenzyme A hydratase/isomerase superfamily based on a common active site template. Biochemistry 38, 7638–7652 (1999)

    Article  CAS  Google Scholar 

  12. Berglund, G. I. et al. The catalytic pathway of horseradish peroxidase at high resolution. Nature 417, 463–468 (2002)

    Article  ADS  CAS  Google Scholar 

  13. Karlsson, A. et al. Crystal structure of naphthalene dioxygenase: side-on binding of dioxygen to iron. Science 299, 1039–1042 (2003)

    Article  ADS  CAS  Google Scholar 

  14. Wentworth, P. et al. Antibody catalysis of the oxidation of water. Science 293, 1806–1811 (2001)

    Article  ADS  CAS  Google Scholar 

  15. Adam, W., Alzerreca, A., Liu, J. C. & Yany, F. Cyclic peroxide 52. alpha-peroxylactones via dehydrative cyclization of alpha-hydroperoxy acids. J. Am. Chem. Soc. 99, 5768–5773 (1977)

    Article  CAS  Google Scholar 

  16. Koo, J. A., Schmidt, S. P. & Schuster, G. B. Bioluminescence of the firefly: key steps in the formation of the electronically excited state for model systems. Proc. Natl Acad. Sci. USA 75, 30–33 (1978)

    Article  ADS  CAS  Google Scholar 

  17. Fetzner, S. Oxygenases without requirement for cofactors or metal ions. Appl. Microbiol. Biotechnol. 60, 243–257 (2002)

    Article  CAS  Google Scholar 

  18. Sciara, G. et al. The structure of ActVA-Orf6, a novel type of monooxygenase involved in actinorhodin biosynthesis. EMBO J. 22, 205–215 (2003)

    Article  CAS  Google Scholar 

  19. Colloc'h, N. et al. Crystal structure of the protein drug urate oxidase-inhibitor complex at 2.05 A resolution. Nature Struct. Biol. 4, 947–952 (1997)

    Article  CAS  Google Scholar 

  20. Beinker, P. et al. Crystal structures of SnoaL2 and AclR: two putative hydroxylases in the biosynthesis of aromatic polyketide antibiotics. J. Mol. Biol. 359, 728–740 (2006)

    Article  CAS  Google Scholar 

  21. Frerichs-Deeken, U. et al. Dioxygenases without requirement for cofactors and their chemical model reaction: compulsory order ternary complex mechanism of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase involving general base catalysis by histidine 251 and single-electron oxidation of the substrate dianion. Biochemistry 43, 14485–14499 (2004)

    Article  CAS  Google Scholar 

  22. Massey, V. Activation of molecular oxygen by flavins and flavoproteins. J. Biol. Chem. 269, 22459–22462 (1994)

    CAS  PubMed  Google Scholar 

  23. Roth, J. P. & Klinman, J. P. Catalysis of electron transfer during activation of O2 by the flavoprotein glucose oxidase. Proc. Natl Acad. Sci. USA 100, 62–67 (2003)

    Article  ADS  CAS  Google Scholar 

  24. Pootoolal, J. et al. Assembling the glycopeptide antibiotic scaffold: The biosynthesis of A47934 from Streptomyces toyocaensis NRRL15009. Proc. Natl Acad. Sci. USA 99, 8962–8967 (2002)

    Article  ADS  CAS  Google Scholar 

  25. Otwinowski, Z. & Minor, W. in Methods in Enzymology: Macromolecular Crystallography Vol. 276 (eds Sweet, R. M. & Carter, C. W.) Part A 307–326 (Academic Press, New York, 1997)

    Book  Google Scholar 

  26. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  Google Scholar 

  27. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, (Pt 12 Pt 1)2126–2132 (2004)

    Article  Google Scholar 

  28. Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  CAS  Google Scholar 

  29. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

Download references

Acknowledgements

We are grateful to A. Heroux and the staff at the Brookhaven National Synchrotron Light Source PXRR for assistance with X-ray data collection and processing. We are grateful to C. C. Tseng, R. S. Klausen and C. T. Walsh for discussions and experimental assistance. We thank P. D. Fortin and A. R. Howard-Jones for assistance in oxygen-binding measurements, Z. Dauter for advice on X-ray data analysis, and members of the Bruner research group and Boston College Chemistry Department for helpful discussions and comments on the manuscript. This work was supported with funds from Boston College and the Damon Runyon Cancer Research Foundation.

Author Contributions P.F.W. crystallized and solved the structure of apo-DpgC. E.N.F. crystallized the DpgC/inhibitor co-complex and with P.F.W. solved the structure. E.N.F constructed and with P.F.W. performed biochemical assays on mutant DpgC enzymes. Y.L. synthesized and biochemically evaluated the inhibitor, DPA-NH-CoA. S.D.B. wrote the manuscript and all authors discussed the results and commented on the manuscript.

The atomic coordinates for the DpgC/inhibitor complex structure have been deposited in the Protein Data Bank with accession code 2NP9.

Author information

Author notes

  1. Paul F. Widboom and Elisha N. Fielding: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA,

    Paul F. Widboom, Elisha N. Fielding, Ye Liu & Steven D. Bruner

Authors
  1. Paul F. Widboom
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elisha N. Fielding
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ye Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Steven D. Bruner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Steven D. Bruner.

Ethics declarations

Competing interests

Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures S1–S4, Supplementary Table 1 and Supplementary Methods. The Supplementary Figures illustrate additional electron density maps, plots of kinetic data characterizing the synthetic inhibitor and mutant enzymes, experimental data for determination of multimeric state and a plot of data from O2 kinetic binding experiments. A Supplementary Table is included with crystallographic statistics. The Supplementary Methods section describes the cloning and purification of DpgC, enzyme kinetic assays, site-directed mutagenesis, molecular weight determination and synthesis of the inhibitor DPA-NH-CoA. (PDF 1123 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Widboom, P., Fielding, E., Liu, Y. et al. Structural basis for cofactor-independent dioxygenation in vancomycin biosynthesis. Nature 447, 342–345 (2007). https://doi.org/10.1038/nature05702

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature05702

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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