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:

13C NMR snapshots of the complex reaction coordinate of pyridoxal phosphate synthase

Nature Chemical Biology volume 4, pages 425–430 (2008)Cite this article

Abstract

The predominant biosynthetic route to vitamin B6 is catalyzed by a single enzyme. The synthase subunit of this enzyme, Pdx1, operates in concert with the glutaminase subunit, Pdx2, to catalyze the complex condensation of ribose 5-phosphate, glutamine and glyceraldehyde 3-phosphate to form pyridoxal 5′-phosphate, the active form of vitamin B6. In previous studies it became clear that many if not all of the reaction intermediates were covalently bound to the synthase subunit, thus making them difficult to isolate and characterize. Here we show that it is possible to follow a single turnover reaction by heteronuclear NMR using 13C- and 15N-labeled substrates as well as 15N-labeled synthase. By denaturing the enzyme at points along the reaction coordinate, we solved the structures of three covalently bound intermediates. This analysis revealed a new 1,5 migration of the lysine amine linking the intermediate to the enzyme during the conversion of ribose 5-phosphate to pyridoxal 5′-phosphate.

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: 13C analysis of covalently bound Pdx1 intermediates.
Figure 2: Summary of 13C chemical shift assignments.
Figure 3: 13C spectra of intermediates bound to 15N-labeled Pdx1.

Similar content being viewed by others

References

  1. Eliot, A.C. & Kirsch, J.F. Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations. Annu. Rev. Biochem. 73, 383–415 (2004).

    Article  CAS  Google Scholar 

  2. Percudani, R. & Peracchi, A. A genomic overview of pyridoxal-phosphate-dependent enzymes. EMBO Rep. 4, 850–854 (2003).

    Article  CAS  Google Scholar 

  3. Amadasi, A. et al. Pyridoxal 5′-phosphate enzymes as targets for therapeutic agents. Curr. Med. Chem. 14, 1291–1324 (2007).

    Article  CAS  Google Scholar 

  4. Bilski, P., Li, M.Y., Ehrenshaft, M., Daub, M.E. & Chignell, C.F. Vitamin B6 (pyridoxine) and its derivatives are efficient singlet oxygen quenchers and potential fungal antioxidants. Photochem. Photobiol. 71, 129–134 (2000).

    Article  CAS  Google Scholar 

  5. Daub, M.E. & Ehrenshaft, M. The photoactivated cercospora toxin cercosporin: contributions to plant disease and fundamental biology. Annu. Rev. Phytopathol. 38, 461–490 (2000).

    Article  CAS  Google Scholar 

  6. Garrido-Franco, M. Pyridoxine 5′-phosphate synthase: de novo synthesis of vitamin B6 and beyond. Biochim. Biophys. Acta 1647, 92–97 (2003).

    Article  CAS  Google Scholar 

  7. Fitzpatrick, T.B. et al. Two independent routes of de novo vitamin B6 biosynthesis: not that different after all. Biochem. J. 407, 1–13 (2007).

    Article  CAS  Google Scholar 

  8. Burns, K.E., Xiang, Y., Kinsland, C.L., McLafferty, F.W. & Begley, T.P. Reconstitution and biochemical characterization of a new pyridoxal-5′-phosphate biosynthetic pathway. J. Am. Chem. Soc. 127, 3682–3683 (2005).

    Article  CAS  Google Scholar 

  9. Raschle, T., Amrhein, N. & Fitzpatrick, T.B. On the two components of pyridoxal 5′-phosphate synthase from Bacillus subtilis. J. Biol. Chem. 280, 32291–32300 (2005).

    Article  CAS  Google Scholar 

  10. Flicker, K. et al. Structural and thermodynamic insights into the assembly of the heteromeric pyridoxal phosphate synthase from Plasmodium falciparum. J. Mol. Biol. 374, 732–748 (2007).

    Article  CAS  Google Scholar 

  11. Tambasco-Studart, M., Tews, I., Amrhein, N. & Fitzpatrick, T.B. Functional analysis of PDX2 from Arabidopsis, a glutaminase involved in vitamin B6 biosynthesis. Plant Physiol. 144, 915–925 (2007).

    Article  CAS  Google Scholar 

  12. Neuwirth, M., Flicker, K., Strohmeier, M., Tews, I. & Macheroux, P. Thermodynamic characterization of the protein-protein interaction in the heteromeric Bacillus subtilis pyridoxalphosphate synthase. Biochemistry 46, 5131–5139 (2007).

    Article  CAS  Google Scholar 

  13. Denslow, S.A., Rueschhoff, E.E. & Daub, M.E. Regulation of the Arabidopsis thaliana vitamin B6 biosynthesis genes by abiotic stress. Plant Physiol. Biochem. 45, 152–161 (2007).

    Article  CAS  Google Scholar 

  14. Tambasco-Studart, M. et al. Vitamin B6 biosynthesis in higher plants. Proc. Natl. Acad. Sci. USA 102, 13687–13692 (2005).

    Article  CAS  Google Scholar 

  15. Wrenger, C., Eschbach, M.L., Muller, I.B., Warnecke, D. & Walter, R.D. Analysis of the vitamin B6 biosynthesis pathway in the human malaria parasite Plasmodium falciparum. J. Biol. Chem. 280, 5242–5248 (2005).

    Article  CAS  Google Scholar 

  16. Wetzel, D.K., Ehrenshaft, M., Denslow, S.A. & Daub, M.E. Functional complementation between the PDX1 vitamin B6 biosynthetic gene of Cercospora nicotianae and pdxJ of Escherichia coli. FEBS Lett. 564, 143–146 (2004).

    Article  CAS  Google Scholar 

  17. Dong, Y.X., Sueda, S., Nikawa, J. & Kondo, H. Characterization of the products of the genes SNO1 and SNZ1 involved in pyridoxine synthesis in Saccharomyces cerevisiae. Eur. J. Biochem. 271, 745–752 (2004).

    Article  CAS  Google Scholar 

  18. Ehrenshaft, M. & Daub, M.E. Isolation of PDX2, a second novel gene in the pyridoxine biosynthesis pathway of eukaryotes, archaebacteria, and a subset of eubacteria. J. Bacteriol. 183, 3383–3390 (2001).

    Article  CAS  Google Scholar 

  19. Strohmeier, M. et al. Structure of a bacterial pyridoxal 5′-phosphate synthase complex. Proc. Natl. Acad. Sci. USA 103, 19284–19289 (2006).

    Article  CAS  Google Scholar 

  20. Zein, F. et al. Structural insights into the mechanism of the PLP synthase holoenzyme from Thermotoga maritima. Biochemistry 45, 14609–14620 (2006).

    Article  CAS  Google Scholar 

  21. Raschle, T. et al. Reaction mechanism of pyridoxal 5′-phosphate synthase. Detection of an enzyme-bound chromophoric intermediate. J. Biol. Chem. 282, 6098–6105 (2007).

    Article  CAS  Google Scholar 

  22. Hanes, J.W., Keresztes, I. & Begley, T.P. Trapping of a chromophoric intermediate in the Pdx1-catalyzed biosynthesis of pyridoxal 5′-phosphate. Angew. Chem. Int. Edn Engl. 47, 2102–2105 (2008).

    Article  CAS  Google Scholar 

  23. Hanes, J.W. et al. Mechanistic studies on pyridoxal phosphate synthase: the reaction pathway leading to a chromophoric intermediate. J. Am. Chem. Soc. 130, 3043–3052 (2008).

    Article  CAS  Google Scholar 

  24. Anderson, K.S. et al. Observation by 13C NMR of the EPSP synthase tetrahedral intermediate bound to the enzyme active site. Biochemistry 29, 1460–1465 (1990).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This research was supported by grants from the US National Institutes of Health to T.P.B. (GM069618).

Author information

Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Cornell University, 120 Baker Laboratory, Ithaca, 14853, New York, USA

    Jeremiah W Hanes, Ivan Keresztes & Tadhg P Begley

Authors
  1. Jeremiah W Hanes
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ivan Keresztes
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tadhg P Begley
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tadhg P Begley.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 and Supplementary Methods (PDF 1192 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hanes, J., Keresztes, I. & Begley, T. 13C NMR snapshots of the complex reaction coordinate of pyridoxal phosphate synthase. Nat Chem Biol 4, 425–430 (2008). https://doi.org/10.1038/nchembio.93

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.93

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