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The catalytic cycle of a thiamin diphosphate enzyme examined by cryocrystallography

Nature Chemical Biology volume 2, pages 324–328 (2006)Cite this article

An Erratum to this article was published on 01 July 2006

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Abstract

Enzymes that use the cofactor thiamin diphosphate (ThDP, 1), the biologically active form of vitamin B1, are involved in numerous metabolic pathways in all organisms. Although a theory of the cofactor's underlying reaction mechanism has been established over the last five decades1,2, the three-dimensional structures of most major reaction intermediates of ThDP enzymes have remained elusive. Here, we report the X-ray structures of key intermediates in the oxidative decarboxylation of pyruvate, a central reaction in carbon metabolism catalyzed by the ThDP- and flavin-dependent enzyme pyruvate oxidase (POX)3 from Lactobacillus plantarum. The structures of 2-lactyl-ThDP (LThDP, 2) and its stable phosphonate analog, of 2-hydroxyethyl-ThDP (HEThDP, 3) enamine and of 2-acetyl-ThDP (AcThDP, 4; all shown bound to the enzyme's active site) provide profound insights into the chemical mechanisms and the stereochemical course of thiamin catalysis. These snapshots also suggest a mechanism for a phosphate-linked acyl transfer coupled to electron transfer in a radical reaction of pyruvate oxidase.

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Figure 1: Detection and quantification of covalent ThDP intermediates in POX catalysis by 1H NMR spectroscopy after acid-quench isolation according to5,12.
Figure 2: Stereo view of the active site of pyruvate oxidase F479W with the two neighboring cofactors ThDP and FAD and the amino acid residues in close proximity to the reactive C2 carbon atom of ThDP.
Figure 3: The four reaction intermediates of the cofactor ThDP in pyruvate oxidase.
Figure 4: Exo-site binding cavity of the substrate pyruvate in the structure of the LThDP-containing enzyme.

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  • 30 May 2006

    supp info PDFs 1 and 2 replaced; compound numbering fixed; notes added to HTML

Notes

  1. *Note: In the supplementary information initially published online to accompany this letter, the legends of Supplementary Figures 1 and 2 are incorrect. The legends contain incorrect citations to other figures; Supplementary Figure 1 should cite Fig. 3a and Supplementary Figure 2 should cite Supplementary Fig. 3. These errors have been corrected online. Also, in the version of this article initially published online, compounds in the Compound Data Index are listed in the wrong order; thus the numbered compounds in the article link to the wrong structures. This error has been corrected in the HTML version of the article.**Note: In the version of this article initially published, there was an error in the text of the second page. In line 15 of the first column, the text should read "a difference Fourier" rather than "a distance Fourier." The error has been corrected in the PDF version of the article.

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Acknowledgements

We gratefully acknowledge access to synchrotron radiation beamlines X13 and BW7B at the European Molecular Biology Laboratory outstation, Deutsches Elektronen-Synchrotron, Hamburg. We thank R. Schowen, R. Kluger, S. Ghisla, F. Jordan and in particular G. Hübner for many stimulating discussions.

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  1. Institut für Biochemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 3, Halle/Saale, 06120, Germany

    Georg Wille, Danilo Meyer, Andrea Steinmetz, Erik Hinze, Ralph Golbik & Kai Tittmann

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  1. Georg Wille
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Correspondence to Georg Wille or Kai Tittmann.

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Wille, G., Meyer, D., Steinmetz, A. et al. The catalytic cycle of a thiamin diphosphate enzyme examined by cryocrystallography. Nat Chem Biol 2, 324–328 (2006). https://doi.org/10.1038/nchembio788

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