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.

Sub-ångström-resolution crystallography reveals physical distortions that enhance reactivity of a covalent enzymatic intermediate

Nature Chemistry volume 5, pages 762–767 (2013)Cite this article

Subjects

Abstract

It is recognized widely that enzymes promote reactions by providing a pathway that proceeds through a transition state of lower energy. In principle, further rate enhancements could be achieved if intermediates are prevented from relaxing to their lowest energy state, and thereby reduce the barrier to the subsequent transition state. Here, we report sub-ångström-resolution crystal structures of genuine covalent reaction intermediates of transketolase. These structures reveal a pronounced out-of-plane distortion of over 20° for the covalent bond that links cofactor and substrate, and a specific elongation of the scissile substrate carbon–carbon bond (d > 1.6 Å). To achieve these distortions, the protein's conformation appears to prevent relaxation of a substrate–cofactor intermediate. The results implicate a reduced barrier to the subsequent step that is consistent with an intermediate of raised energy and leads to a more efficient overall process.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: TK reactions.
Figure 2: Structural evidence for physically distorted covalent intermediates in human TK.
Figure 3: Origin of strain in the covalent X5P–ThDP conjugate bound to human TK.
Figure 4: Principles of enzyme catalysis.

Accession codes

Primary accessions

Protein Data Bank

References

  1. Pauling, L., Chemical achievement and hope for the future. Am. Sci. 36, 50–58 (1948).

    Google Scholar 

  2. Radzicka, A. & Wolfenden, R., A proficient enzyme. Science 267, 90–93 (1995).

    Article  CAS  Google Scholar 

  3. Albery, W. J. & Knowles, J. R. Efficiency and evolution of enzyme catalysis. Angew. Chem. Int. Ed. Engl. 16, 285–293 (1977).

    Article  CAS  Google Scholar 

  4. Wu, N., Mo, Y. R., Gao, J. L. & Pai, E. F. Electrostatic stress in catalysis: structure and mechanism of the enzyme orotidine monophosphate decarboxylase. Proc. Natl Acad. Sci. USA 97, 2017–2022 (2000).

    Article  CAS  Google Scholar 

  5. Amyes, T. L. & Richard, J. P. Specificity in transition state binding: the Pauling model revisited. Biochemistry 52, 2021–2035 (2013).

    Article  CAS  Google Scholar 

  6. Asztalos, P. et al. Strain and near attack conformers in enzymic thiamin catalysis: X-ray crystallographic snapshots of bacterial transketolase in covalent complex with donor ketoses xylulose 5-phosphate and fructose 6-phosphate, and in noncovalent complex with acceptor aldose ribose 5-phosphate. Biochemistry 46, 12037–12052 (2007).

    Article  CAS  Google Scholar 

  7. Lehwess-Litzmann, A. et al. Twisted Schiff base intermediates and substrate locale revise transaldolase mechanism. Nature Chem. Biol. 7, 678–684 (2011).

    Article  CAS  Google Scholar 

  8. Milic, D. et al. Crystallographic snapshots of tyrosine phenol-lyase show that substrate strain plays a role in C–C bond cleavage. J. Am. Chem. Soc. 133, 16468–16476 (2011).

    Article  CAS  Google Scholar 

  9. Mitschke, L. et al. The crystal structure of human transketolase and new insights into its mode of action. J. Biol. Chem. 285, 31559–31570 (2010).

    Article  CAS  Google Scholar 

  10. Schenk, G., Duggleby, R. G. & Nixon, P F. Properties and functions of the thiamin diphosphate dependent enzyme transketolase. Int. J. Biochem. Cell Biol. 30, 1297–1318 (1998).

    Article  CAS  Google Scholar 

  11. Tittmann, K. et al. NMR analysis of covalent intermediates in thiamin diphosphate enzymes. Biochemistry 42, 7885–7891 (2003).

    Article  CAS  Google Scholar 

  12. Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008).

    Article  CAS  Google Scholar 

  13. Arjunan, P. et al. A thiamin-bound, pre-decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1 subunit induces large scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound adduct. J. Biol. Chem. 281, 15296–15303 (2006).

    Article  CAS  Google Scholar 

  14. Wille, G. et al. The catalytic cycle of a thiamin diphosphate enzyme examined by cryocrystallography. Nature Chem. Biol. 2, 324–328 (2006).

    Article  CAS  Google Scholar 

  15. Fiedler, E. et al. Snapshot of a key intermediate in enzymatic thiamin catalysis: crystal structure of the alpha-carbanion of (α,β-dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA 99, 591–595 (2002).

    Article  CAS  Google Scholar 

  16. Leeper, F. J. & Smith, D. H. C. The effect of strain on the catalytic reactions of bridged thiazolium salts as models of thiamin. J. Chem. Soc. Chem. Commun. 14, 961–963 (1990).

    Article  Google Scholar 

  17. Turano, A. et al. Synthesis and crystal-structure of an analog of 2-(alpha-lactyl)thiamin, racemic methyl 2-hydroxy-2-(2-thiamin)ethylphosphonate chloride trihydrate – a conformation for a least-motion, maximum-overlap mechanism for thiamin catalysis. J. Am. Chem. Soc. 104, 3089–3095 (1982).

    Article  CAS  Google Scholar 

  18. Kluger, R. & Tittmann, K. Thiamin diphosphate catalysis: enzymic and nonenzymic covalent intermediates. Chem. Rev. 108, 1797–1833 (2008).

    Article  CAS  Google Scholar 

  19. Jeffrey, G. A. & Kim, H. S. Conformations of alditols. Carbohyd. Res. 14, 207–216 (1970).

    Article  CAS  Google Scholar 

  20. Allen, F. H. et al. Tables of bond lengths determined by X-ray and neutron-diffraction. 1. Bond lengths in organic-compounds. J. Chem. Soc. Perk. Trans. 2 12, S1–S19 (1987).

    Google Scholar 

  21. Zavitsas, A. A. The relation between bond lengths and dissociation energies of carbon–carbon bonds. J. Phys. Chem. A 107, 897–898 (2003).

    Article  CAS  Google Scholar 

  22. Kaupp, G. & Boy, J. Overlong C–C single bonds. Angew. Chem. Int. Ed. Engl. 36, 48–49 (1997).

    Article  CAS  Google Scholar 

  23. Toda, F. Naphthocyclobutenes and benzodicyclobutadienes: synthesis in the solid state and anomalies in the bond lengths. Eur. J. Org. Chem. 8, 1377–1386 (2000).

    Article  Google Scholar 

  24. Schreiner, P. R. et al. Overcoming lability of extremely long alkane carbon–carbon bonds through dispersion forces. Nature 477, 308–311 (2011).

    Article  CAS  Google Scholar 

  25. Nemeria, N. et al. Tetrahedral intermediates in thiamin diphosphate-dependent decarboxylations exist as a 1′,4′-imino tautomeric form of the coenzyme, unlike the Michaelis complex or the free coenzyme. Biochemistry 43, 6565–6575 (2004).

    Article  CAS  Google Scholar 

  26. Nemeria, N. et al. The 1′,4′-iminopyrimidine tautomer of thiamin diphosphate is poised for catalysis in asymmetric active centers on enzymes. Proc. Natl Acad. Sci. USA 104, 78–82 (2007).

    Article  CAS  Google Scholar 

  27. Schowen, R. L. in Comprehensive Biological Catalysis Vol. 2 (ed. Sinnott M. L. ) 217–266 (Academic Press, 1998).

  28. Jencks, W. P. Binding-energy, specificity, and enzymic catalysis – Circe effect. Adv. Enzym. Rel. Areas Mol. Biol. 43, 219–410 (1975).

    CAS  Google Scholar 

  29. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  31. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 5, 1948–1954 (2002).

    Article  Google Scholar 

  32. Brunger, A. T. Free R-value – a novel statistical quantity for assessing the accuracy of crystal structures. Nature 355, 472–475 (1992).

    Article  CAS  Google Scholar 

  33. Thorn, A., Dittrich, B. & Sheldrick, G. M. Enhanced rigid-bond restraints. Acta Cryst. A 68, 448–451 (2012).

    Article  CAS  Google Scholar 

  34. Wang, J. J., Martin, P. R. & Singleton, C. K. Aspartate 155 of human transketolase is essential for thiamine diphosphate–magnesium binding, and cofactor binding is required for dimer formation. Biochim. Biophys. Acta 134, 165–172 (1997).

    Article  Google Scholar 

  35. Hawksley, D., Griffin, D. A. & Leeper, F. J. Synthesis of 3-deazathiamine. J. Chem. Soc. Perkin Trans. 1, 144–148 (2001).

    Article  Google Scholar 

  36. Thomas, A. A. et al. Non-charged thiamine analogs as inhibitors of enzyme transketolase. Bioorg. Med. Chem. Lett. 18, 509–512 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank G. M. Sheldrick for providing a pre-release version of software SHELXL-2012. We are grateful for synchrotron beam time and local support at the ESRF Grenoble, France, and at the BESSY Berlin, Germany. This work was supported through grants of the Fonds der Chemischen Industrie (K.T.), Forschergruppe 1296 funded by the Deutsche Forschungsgemeinschaft (K.T.) and the Sonderforschungsbereich 860 funded by the Deutsche Forschungsgemeinschaft (R.F.).

Author information

Authors and Affiliations

  1. Albrecht-von-Haller Institute, Göttingen Center for Molecular Biosciences, Georg-August University Göttingen, Göttingen, 37077, Germany

    Stefan Lüdtke & Kai Tittmann

  2. Institute of Microbiology and Genetics, Göttingen Center for Molecular Biosciences, Georg-August University Göttingen, Göttingen, 37077, Germany

    Piotr Neumann & Ralf Ficner

  3. Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK

    Karl M. Erixon & Finian Leeper

  4. Department of Chemistry, Davenport Chemical Laboratories, University of Toronto, Ontario, M5S 3H6, Canada

    Ronald Kluger

Authors
  1. Stefan Lüdtke
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Piotr Neumann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Karl M. Erixon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Finian Leeper
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ronald Kluger
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ralf Ficner
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kai Tittmann
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.T. conceived and supervised the project; S.L. expressed, purified and crystallized the enzyme; S.L. and P.N. collected data sets at ESFR Grenoble and BESSY Berlin; S.L. refined structures; P.N. conducted final refinement rounds and e.s.d. values using the software SHELXL-2012; S.L. conducted CD measurements; K.M.E. and F.L. synthesized the thiamin analogue; K.T., S.L., P.N., K.M.E., F.L., R.F. and R.K. discussed the data. R.K. and K.T. co-wrote the paper with input from all other authors. S.L. and P.N. contributed equally to this study.

Corresponding author

Correspondence to Kai Tittmann.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 1948 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lüdtke, S., Neumann, P., Erixon, K. et al. Sub-ångström-resolution crystallography reveals physical distortions that enhance reactivity of a covalent enzymatic intermediate. Nature Chem 5, 762–767 (2013). https://doi.org/10.1038/nchem.1728

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchem.1728

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