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Twisted Schiff base intermediates and substrate locale revise transaldolase mechanism

Nature Chemical Biology volume 7, pages 678684 (2011) | Download Citation

Abstract

We examined the catalytic cycle of transaldolase (TAL) from Thermoplasma acidophilum by cryocrystallography and were able to structurally characterize—for the first time, to our knowledge—different genuine TAL reaction intermediates. These include the Schiff base adducts formed between the catalytic lysine and the donor ketose substrates fructose-6-phosphate and sedoheptulose-7-phosphate as well as the Michaelis complex with acceptor aldose erythrose-4-phosphate. These structural snapshots necessitate a revision of the accepted reaction mechanism with respect to functional roles of active site residues, and they further reveal fundamental insights into the general structural features of enzymatic Schiff base intermediates and the role of conformational dynamics in enzyme catalysis, substrate binding and discrimination. A nonplanar arrangement of the substituents around the Schiff base double bond was observed, suggesting that a structurally encoded reactant-state destabilization is a driving force of catalysis. Protein dynamics and the intrinsic hydrogen-bonding pattern appear to be crucial for selective recognition and binding of ketose as first substrate.

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References

  1. 1.

    & Transaldolase: from biochemistry to human disease. Int. J. Biochem. Cell Biol. 41, 1482–1494 (2009).

  2. 2.

    The pentose phosphate pathway. J. Biol. Chem. 277, 47965–47971 (2002).

  3. 3.

    , & Identity of synthetic N6-beta-glyceryllysine and C14-labeled amino acid obtained on sodium borohydride reduction and hydrolysis of a complex from C14-fructose 6-phosphate- transaldolase interaction. J. Am. Chem. Soc. 85, 1012–1013 (1963).

  4. 4.

    et al. Crystal structure of transaldolase B from Escherichia coli suggests a circular permutation of the alpha/beta barrel within the class I aldolase family. Structure 4, 715–724 (1996).

  5. 5.

    , , , & The three-dimensional structure of human transaldolase. FEBS Lett. 475, 205–208 (2000).

  6. 6.

    , , , & Crystal structure of the reduced Schiff-base intermediate complex of transaldolase B from Escherichia coli: mechanistic implications for class I aldolases. Protein Sci. 6, 119–124 (1997).

  7. 7.

    et al. Identification of catalytically important residues in the active site of Escherichia coli transaldolase. Eur. J. Biochem. 268, 2408–2415 (2001).

  8. 8.

    , , , & Crystallization and preliminary X-ray diffraction analysis of transaldolase from Thermoplasma acidophilum. Acta. Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 67, 584–586 (2011).

  9. 9.

    , , & Crystal structure of decameric fructose-6-phosphate aldolase from Escherichia coli reveals inter-subunit helix swapping as a structural basis for assembly differences in the transaldolase family. J. Mol. Biol. 319, 161–171 (2002).

  10. 10.

    , , , & Replacement of a phenylalanine by a tyrosine in the active site confers fructose-6-phosphate aldolase activity to the transaldolase of Escherichia coli and human origin. J. Biol. Chem. 283, 30064–30072 (2008).

  11. 11.

    , & Conformations of acyclic sugar derivatives: part II. Determination of conformations of alditol acetates in solution by use of 250-Mhz n.m.r. spectra. Carbohydr. Res. 23, 121–134 (1972).

  12. 12.

    , & Carbon-13-enriched carbohydrates. Preparation of erythrose, threose, glyceraldehyde, and glycolaldehyde with 13C enrichment in various carbon atoms. Carbohydr. Res. 72, 79–91 (1979).

  13. 13.

    , , & High resolution reaction intermediates of rabbit muscle fructose-1,6-bisphosphate aldolase: substrate cleavage and induced fit. J. Biol. Chem. 280, 27262–27270 (2005).

  14. 14.

    , & Very fast prediction and rationalization of pKa values for protein–ligand complexes. Proteins. 73, 765–783 (2008).

  15. 15.

    et al. Redesigning the active site of transaldolase TalB from Escherichia coli: new variants with improved affinity towards nonphosphorylated substrates. ChemBioChem 11, 681–690 (2010).

  16. 16.

    , & Structure of a class I tagatose-1,6-bisphosphate aldolase investigation into an apparent loss of stereospecificity. J. Biol. Chem. 285, 21143–21152 (2010).

  17. 17.

    & Efficiency and evolution of enzyme catalysis. Angew. Chem. Int. Edn Engl. 16, 285–293 (1977).

  18. 18.

    et al. Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289, 739–745 (2000).

  19. 19.

    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).

  20. 20.

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

  21. 21.

    , & The role of dynamic conformational ensembles in biomolecular recognition. Nat. Chem. Biol. 5, 789–796 (2009).

  22. 22.

    , & Conformational selection or induced fit: a flux description of reaction mechanism. Proc. Natl. Acad. Sci. USA 106, 13737–13741 (2009).

  23. 23.

    , , & Preparative scale enzymatic synthesis of D-sedoheptulose-7-phosphate from β-hydroxypyruvate and D-ribose-5-phosphate. J. Mol. Catal. B Enzym. 57, 6–9 (2009).

  24. 24.

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

  25. 25.

    , & Dimerization of erythrose 4-phosphate. FEBS Lett. 64, 222–226 (1976).

  26. 26.

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

  27. 27.

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

  28. 28.

    & Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

  29. 29.

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

  30. 30.

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

  31. 31.

    , & SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model. Acta Crystallogr. D Biol. Crystallogr. 55, 191–205 (1999).

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Acknowledgements

We gratefully acknowledge access to synchrotron radiation beamtime at the Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung (BESSY). This work was supported by the Friedrich-Naumann-Stiftung (stipend to A.L.-L.), the Fonds der Chemischen Industrie (stipend to S.L.), and the Göttingen Graduate School for Neurosciences and Molecular Biosciences funded by the Deutsche Forschungsgemeinschaft (to K.T.).

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Affiliations

  1. Albrecht-von-Haller-Institut and Göttingen Center for Molecular Biosciences, Georg-August-Universität Göttingen, Göttingen, Germany.

    • Anja Lehwess-Litzmann
    • , Stefan Lüdtke
    •  & Kai Tittmann
  2. Institute of Microbiology and Genetics, Georg-August-Universität, Göttingen, Germany.

    • Piotr Neumann
    •  & Ralf Ficner
  3. Institute of Biochemistry and Biotechnology, Martin-Luther-Universität Halle-Wittenberg, Halle, Germany.

    • Christoph Parthier
    •  & Ralph Golbik

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Contributions

K.T. designed the study; A.L.-L. and R.G. recombinantly expressed and purified TAL; A.L.-L. crystallized TAL; A.L.-L., P.N. and C.P. recorded the diffraction data; A.L.-L. and P.N. solved the structures; A.L.-L. refined the structures; S.L. chemoenzymatically synthesized ketose substrate S7P; A.L.-L., P.N., C.P., R.F. and K.T. discussed the data; and K.T. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Kai Tittmann.

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https://doi.org/10.1038/nchembio.633

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