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:

Glyoxylate carboligase lacks the canonical active site glutamate of thiamine-dependent enzymes

Nature Chemical Biology volume 4, pages 113–118 (2008)Cite this article

Abstract

Thiamine diphosphate (ThDP), a derivative of vitamin B1, is an enzymatic cofactor whose special chemical properties allow it to play critical mechanistic roles in a number of essential metabolic enzymes. It has been assumed that all ThDP-dependent enzymes exploit a polar interaction between a strictly conserved glutamate and the N1′ of the ThDP moiety. The crystal structure of glyoxylate carboligase challenges this paradigm by revealing that valine replaces the conserved glutamate. Through kinetic, spectroscopic and site-directed mutagenesis studies, we show that although this extreme change lowers the rate of the initial step of the enzymatic reaction, it ensures efficient progress through subsequent steps. Glyoxylate carboligase thus provides a unique illustration of the fine tuning between catalytic stages imposed during evolution on enzymes catalyzing multistep processes.

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: GCL reaction and the role of the conserved glutamate in the mechanism of ThDP-dependent enzymes.
Figure 2: Structure of GCL monomer and details of ThDP binding site.
Figure 3: Cofactor reactivity in GCL wild type compared with variants at Val51.
Figure 4: Steady state intermediate distribution of GCL wild type and of variants V51D and V51E.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Muller, Y.A. et al. A thiamin diphosphate binding fold revealed by comparison of the crystal structures of transketolase, pyruvate oxidase and pyruvate decarboxylase. Structure 1, 95–103 (1993).

    Article  CAS  Google Scholar 

  2. Kern, D. et al. How thiamine diphosphate is activated in enzymes. Science 275, 67–70 (1997).

    Article  CAS  Google Scholar 

  3. Breslow, R. On the mechanism of thiamine action: IV. Evidence from studies on model systems. J. Am. Chem. Soc. 80, 3719–3726 (1958).

    Article  CAS  Google Scholar 

  4. Schellenberger, A. Structure and mechanism of action of the active center of yeast pyruvate decarboxylase. Angew. Chem. Int. Ed. Engl. 6, 1024–1035 (1967).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Gupta, N. & Vennesland, B. Glyoxylate carboligase of Escherichia coli: some properties of the enzyme. Arch. Biochem. Biophys. 113, 255–264 (1966).

    Article  CAS  Google Scholar 

  7. Krakow, G. & Barkulis, S.S. Conversion of glyoxylate to hydroxypyruvate by extracts of Escherichia coli. Biochim. Biophys. Acta 21, 593–594 (1956).

    Article  CAS  Google Scholar 

  8. Gotto, A.M. & Kornberg, H.L. The metabolism of C2 compounds in micro-organisms. 7. Preparation and properties of crystalline tartronic semialdehyde reductase. Biochem. J. 81, 273–284 (1961).

    Article  CAS  Google Scholar 

  9. Vinogradov, M. et al. Monitoring the acetohydroxy acid synthase reaction and related carboligations by circular dichroism spectroscopy. Anal. Biochem. 342, 126–133 (2005).

    Article  CAS  Google Scholar 

  10. Krakow, G., Barkulis, S.S. & Hayashi, J.A. Glyoxylic acid carboligase: an enzyme present in glycolate-grown Escherichia coli. J. Bacteriol. 81, 509–518 (1961).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Van der Drift, C. & De Windt, F.E. Glyoxylate conversion by Hyphomicrobium species grown on allantoin as nitrogen source. Antonie Van Leeuwenhoek 49, 167–172 (1983).

    Article  CAS  Google Scholar 

  12. 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 

  13. Hendrickson, W.A. Determination of macromolecular structures from anomalous diffraction of synchrotron radiation. Science 254, 51–58 (1991).

    Article  CAS  Google Scholar 

  14. Duggleby, R.G. Domain relationships in thiamine diphosphate-dependent enzymes. Acc. Chem. Res. 39, 550–557 (2006).

    Article  CAS  Google Scholar 

  15. Mosbacher, T.G., Mueller, M. & Schulz, G.E. Structure and mechanism of the ThDP-dependent benzaldehyde lyase from Pseudomonas fluorescens. FEBS J. 272, 6067–6076 (2005).

    Article  CAS  Google Scholar 

  16. Dyda, F. et al. Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4 Å resolution. Biochemistry 32, 6165–6170 (1993).

    Article  CAS  Google Scholar 

  17. Lindqvist, Y. & Schneider, G. Thiamin diphosphate dependent enzymes: transketolase, pyruvate oxidase and pyruvate decarboxylase. Curr. Opin. Struct. Biol. 3, 896–901 (1993).

    Article  CAS  Google Scholar 

  18. Lindqvist, Y., Schneider, G., Ermler, U. & Sundstrom, M. Three-dimensional structure of transketolase, a thiamine diphosphate dependent enzyme, at 2.5 resolution. EMBO J. 11, 2373–2379 (1992).

    Article  CAS  Google Scholar 

  19. Schellenberger, A. Die Funktion der 4′-Aminopyrimidin-Komponente im Katalysemechanismus von Thiamin pyrophosphat-Enzymen aus heutiger Sicht. Chem. Ber. 123, 1489–1494 (1990).

    Article  CAS  Google Scholar 

  20. Bar-Ilan, A. et al. Binding and activation of thiamin diphosphate in acetohydroxyacid synthase. Biochemistry 40, 11946–11954 (2001).

    Article  CAS  Google Scholar 

  21. Golbik, R. et al. Function of the aminopyrimidine part in thiamine pyrophosphate enzymes. Bioorg. Chem. 19, 10–17 (1991).

    Article  CAS  Google Scholar 

  22. 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 

  23. Nemeria, N. et al. Elucidation of the chemistry of enzyme-bound thiamin diphosphate prior to substrate binding: defining internal equilibria among tautomeric and ionization states. Biochemistry 46, 10739–10744 (2007).

    Article  CAS  Google Scholar 

  24. Tittmann, K., Vyazmensky, M., Hubner, G., Barak, Z. & Chipman, D.M. The carboligation reaction of acetohydroxyacid synthase II: steady-state intermediate distributions in wild type and mutants by NMR. Proc. Natl. Acad. Sci. USA 102, 553–558 (2005).

    Article  CAS  Google Scholar 

  25. Gutowski, J.A. & Lienhard, G.E. Transition state analogs for thiamin pyrophosphate-dependent enzymes. J. Biol. Chem. 251, 2863–2866 (1976).

    CAS  PubMed  Google Scholar 

  26. Kemp, D.S. & O'Brien, J.T. Base catalysis of thiazolium salt hydrogen exchange and its implications for enzymatic thiamine cofactor catalysis. J. Am. Chem. Soc. 92, 2554–2555 (1970).

    Article  CAS  Google Scholar 

  27. Zhang, S. et al. Evidence for dramatic acceleration of a C-H bond ionization rate in thiamin diphosphate enzymes by the protein environment. Biochemistry 44, 2237–2243 (2005).

    Article  CAS  Google Scholar 

  28. Zhang, S., Liu, M., Yan, Y., Zhang, Z. & Jordan, F. C2-alpha-lactylthiamin diphosphate is an intermediate on the pathway of thiamin diphosphate-dependent pyruvate decarboxylation. Evidence on enzymes and models. J. Biol. Chem. 279, 54312–54318 (2004).

    Article  CAS  Google Scholar 

  29. Jordan, F., Li, H. & Brown, A. Remarkable stabilization of zwitterionic intermediates may account for a billion-fold rate acceleration by thiamin diphosphate-dependent decarboxylases. Biochemistry 38, 6369–6373 (1999).

    Article  CAS  Google Scholar 

  30. Van Duyne, G.D., Standaert, R.F., Karplus, P.A., Schreiber, S.L. & Clardy, J. Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. J. Mol. Biol. 229, 105–124 (1993).

    Article  CAS  Google Scholar 

  31. Chung, S.T., Tan, R.T. & Suzuki, I. Glyoxylate carboligase of Pseudomonas oxalaticus. A possible structural role for flavine-adenine dinucleotide. Biochemistry 10, 1205–1209 (1971).

    Article  CAS  Google Scholar 

  32. Chang, Y.Y., Wang, A.Y. & Cronan, J.E., Jr. Molecular cloning, DNA sequencing, and biochemical analyses of Escherichia coli glyoxylate carboligase: an enzyme of the acetohydroxy acid synthase-pyruvate oxidase family. J. Biol. Chem. 268, 3911–3919 (1993).

    CAS  PubMed  Google Scholar 

  33. Schloss, J.V., Ciskanik, L.M. & Van Dyk, D.E. Origin of the herbicide binding site of acetolactate synthase. Nature 331, 360–362 (1988).

    Article  CAS  Google Scholar 

  34. 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 

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

  36. McCoy, A., Grosse-Kunstleve, R., Storoni, L. & Read, R. Likelihood-enhanced fast translation functions. Acta Crystallogr. D Biol. Crystallogr. 61, 458–464 (2005).

    Article  Google Scholar 

  37. de la Fortelle, E. & Bricogne, G. Maximum likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–494 (1997).

    Article  CAS  Google Scholar 

  38. Jones, T.A., Zou, J.-Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  Google Scholar 

  39. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  40. Brünger, A. et al. Crystallography and NMR system: a new software for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

    Article  Google Scholar 

  41. Richardson, J.S., Arendall, W.B., III & Richardson, D.C. New tools and data for improving structures, using all-atom contacts. Methods Enzymol. 374, 385–412 (2003).

    Article  CAS  Google Scholar 

  42. Holm, L. & Sander, C. Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993).

    Article  CAS  Google Scholar 

  43. Pettersen, E.F. et al. UCSF Chimera - a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge the ESRF for provision of synchrotron radiation facilities, and we thank G. Leonard for assistance in using beamline ID14-4. This research was partially supported by Israel Science Foundation grants 467/02-1 (to Z.B. and D.M.C.) and 716/06 (to B.S. and D.M.C.), and by grant 126FP/0705M from the Ministry of Education of Saxony-Anhalt (to K.T.). We thank R. Golbik for an authentic sample of N3′-pyridyl-ThDP.

Author information

Authors and Affiliations

  1. Department of Life Sciences, Ben-Gurion University of the Negev, 1 Ben-Gurion Avenue, Beer-Sheva, 84105, Israel

    Alexander Kaplun, Elad Binshtein, Maria Vyazmensky, Ze'ev Barak, David M Chipman & Boaz Shaanan

  2. Martin-Luther-Universität Halle-Wittenberg, Institut für Biochemie, Kurt-Mothes-Strasse 3, Halle, D-06099, Germany

    Andrea Steinmetz & Kai Tittmann

Authors
  1. Alexander Kaplun
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elad Binshtein
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maria Vyazmensky
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andrea Steinmetz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ze'ev Barak
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. David M Chipman
    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

  8. Boaz Shaanan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.K. and Z.B. initiated the work on GCL; A.K. and M.V. cloned GCL; A.K. purified and crystallized GCL; A.K. and B.S. recorded the diffraction data; B.S. solved the structure; E.B. prepared the GCL mutants; E.B., M.V., Z.B. and D.M.C. studied the steady state kinetics; A.S. and K.T. carried out the NMR and CD studies; D.M.C., K.T. and B.S. wrote the paper, with critical suggestions from all other authors.

Corresponding author

Correspondence to Boaz Shaanan.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 1 (PDF 2203 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kaplun, A., Binshtein, E., Vyazmensky, M. et al. Glyoxylate carboligase lacks the canonical active site glutamate of thiamine-dependent enzymes. Nat Chem Biol 4, 113–118 (2008). https://doi.org/10.1038/nchembio.62

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.62

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