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Reconstitution of ThiC in thiamine pyrimidine biosynthesis expands the radical SAM superfamily

Nature Chemical Biology volume 4, pages 758–765 (2008)Cite this article

Abstract

4-Amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase catalyzes a complex rearrangement of 5-aminoimidazole ribonucleotide (AIR) to form HMP-P, the pyrimidine moiety of thiamine phosphate. We determined the three-dimensional structures of HMP-P synthase and its complexes with the product HMP-P and a substrate analog imidazole ribotide. The structure of HMP-P synthase reveals a homodimer in which each protomer comprises three domains: an N-terminal domain with a novel fold, a central (βα)8 barrel and a disordered C-terminal domain that contains a conserved CX2CX4C motif, which is suggestive of a [4Fe-4S] cluster. Biochemical studies have confirmed that HMP-P synthase is iron sulfur cluster–dependent, that it is a new member of the radical SAM superfamily and that HMP-P and 5′-deoxyadenosine are products of the reaction. Mössbauer and EPR spectroscopy confirm the presence of one [4Fe-4S] cluster. Structural comparisons reveal that HMP-P synthase is homologous to a group of adenosylcobalamin radical enzymes. This similarity supports an evolutionary relationship between these two superfamilies.

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Figure 1: The biosynthesis of thiamine pyrophosphate.
Figure 2: HMP-P synthase activity.
Figure 3: EPR and Mössbauer spectroscopy of HMP-P synthase.
Figure 4: Structure of HMP-P synthase.
Figure 5: Cartoons depicting the domain assemblies of cobalamin-dependent enzymes with HMP-P synthase–like protomers and dimer interfaces.

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References

  1. Begley, T.P. et al. Thiamin biosynthesis in prokaryotes. Arch. Microbiol. 171, 293–300 (1999).

    Article  CAS  Google Scholar 

  2. Settembre, E., Begley, T.P. & Ealick, S.E. Structural biology of enzymes of the thiamin biosynthesis pathway. Curr. Opin. Struct. Biol. 13, 739–747 (2003).

    Article  CAS  Google Scholar 

  3. Chatterjee, A., Jurgenson, C.T., Schroeder, F.C., Ealick, S.E. & Begley, T.P. Thiamin biosynthesis in eukaryotes: characterization of the enzyme-bound product of thiazole synthase from Saccharomyces cerevisiae and its implications in thiazole biosynthesis. J. Am. Chem. Soc. 128, 7158–7159 (2006).

    Article  CAS  Google Scholar 

  4. Chatterjee, A., Jurgenson, C.T., Schroeder, F.C., Ealick, S.E. & Begley, T.P. Biosynthesis of thiamin thiazole in eukaryotes: conversion of NAD to an advanced intermediate. J. Am. Chem. Soc. 129, 2914–2922 (2007).

    Article  CAS  Google Scholar 

  5. Jurgenson, C.T., Chatterjee, A., Begley, T.P. & Ealick, S.E. Structural insights into the function of the thiamin biosynthetic enzyme Thi4 from Saccharomyces cerevisiae. Biochemistry 45, 11061–11070 (2006).

    Article  CAS  Google Scholar 

  6. Kriek, M. et al. Thiazole synthase from Escherichia coli: an investigation of the substates and purified proteins required for activity in vitro. J. Biol. Chem. 282, 17413–17423 (2007).

    Article  CAS  Google Scholar 

  7. Lawhorn, B.G., Mehl, R.A. & Begley, T.P. Biosynthesis of the thiamin pyrimidine: the reconstitution of a remarkable rearrangement reaction. Org. Biomol. Chem. 2, 2538–2546 (2004).

    Article  CAS  Google Scholar 

  8. Newell, P.C. & Tucker, R.G. Biosynthesis of the pyrimidine moiety of thiamine. A new route of pyrimidine biosynthesis involving purine intermediates. Biochem. J. 106, 279–287 (1968).

    Article  CAS  Google Scholar 

  9. Zeidler, J., Sayer, B.G. & Spenser, I.D. Biosynthesis of vitamin B1 in yeast. Derivation of the pyrimidine unit from pyridoxine and histidine. Intermediacy of urocanic acid. J. Am. Chem. Soc. 125, 13094–13105 (2003).

    Article  CAS  Google Scholar 

  10. Frey, P.A. & Booker, S.J. Radical mechanisms of S-adenosylmethionine-dependent enzymes. Adv. Protein Chem. 58, 1–45 (2001).

    Article  CAS  Google Scholar 

  11. Wang, S.C. & Frey, P.A. S-adenosylmethionine as an oxidant: the radical SAM superfamily. Trends Biochem. Sci. 32, 101–110 (2007).

    Article  CAS  Google Scholar 

  12. Sofia, H.J., Chen, G., Hetzler, B.G., Reyes-Spindola, J.F. & Miller, N.E. Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods. Nucleic Acids Res. 29, 1097–1106 (2001).

    Article  CAS  Google Scholar 

  13. Dougherty, M.J. & Downs, D.M. A connection between iron-sulfur cluster metabolism and the biosynthesis of 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate in Salmonella enterica. Microbiology 152, 2345–2353 (2006).

    Article  CAS  Google Scholar 

  14. Raschke, M. et al. Vitamin B1 biosynthesis in plants requires the essential iron sulfur cluster protein, THIC. Proc. Natl. Acad. Sci. USA 104, 19637–19642 (2007).

    Article  CAS  Google Scholar 

  15. Reddick, J.J., Nicewonger, R. & Begley, T.P. Mechanistic studies on thiamin phosphate synthase: evidence for a dissociative mechanism. Biochemistry 40, 10095–10102 (2001).

    Article  CAS  Google Scholar 

  16. Park, J.-H., Burns, K., Kinsland, C. & Begley, T.P. Characterization of two kinases involved in thiamine pyrophosphate and pyridoxal phosphate biosynthesis in Bacillus subtilis: 4-amino-5-hydroxymethyl-2-methylpyrimidine kinase and pyridoxal kinase. J. Bacteriol. 186, 1571–1573 (2004).

    Article  CAS  Google Scholar 

  17. Cicchillo, R.M. et al. Escherichia coli lipoyl synthase binds two distinct [4Fe-4S] clusters per polypeptide. Biochemistry 43, 11770–11781 (2004).

    Article  CAS  Google Scholar 

  18. Münck, E. in Physical Methods in Bioinorganic Chemistry (ed. Que, L. Jr.) 287–319 (University Science Books, Sausalito, California, USA, 2000).

    Google Scholar 

  19. Vallazza, M. et al. First look at RNA in L-configuration. Acta Crystallogr. D Biol. Crystallogr. 60, 1–7 (2004).

    Article  CAS  Google Scholar 

  20. Layer, G., Moser, J., Heinz, D.W., Jahn, D. & Schubert, W.D. Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of radical SAM enzymes. EMBO J. 22, 6214–6224 (2003).

    Article  CAS  Google Scholar 

  21. Hanzelmann, P. & Schindelin, H. Crystal structure of the S-adenosylmethionine-dependent enzyme MoaA and its implications for molybdenum cofactor deficiency in humans. Proc. Natl. Acad. Sci. USA 101, 12870–12875 (2004).

    Article  Google Scholar 

  22. Lepore, B.W., Ruzicka, F.J., Frey, P.A. & Ringe, D. The x-ray crystal structure of lysine-2,3-aminomutase from Clostridium subterminale. Proc. Natl. Acad. Sci. USA 102, 13819–13824 (2005).

    Article  CAS  Google Scholar 

  23. Reitzer, R. et al. Glutamate mutase from Clostridium cochlearium: the structure of a coenzyme B12-dependent enzyme provides new mechanistic insights. Structure 7, 891–902 (1999).

    Article  CAS  Google Scholar 

  24. Berkovitch, F. et al. A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase. Proc. Natl. Acad. Sci. USA 101, 15870–15875 (2004).

    Article  CAS  Google Scholar 

  25. Evans, J.C. et al. Structures of the N-terminal modules imply large domain motions during catalysis by methionine synthase. Proc. Natl. Acad. Sci. USA 101, 3729–3736 (2004).

    Article  CAS  Google Scholar 

  26. Mancia, F. et al. How coenzyme B12 radicals are generated: the crystal structure of methylmalonyl-coenzyme A mutase at 2 Å resolution. Structure 4, 339–350 (1996).

    Article  CAS  Google Scholar 

  27. Svetlitchnaia, T., Svetlitchnyi, V., Meyer, O. & Dobbek, H. Structural insights into methyltransfer reactions of a corrinoid iron-sulfur protein involved in acetyl-CoA synthesis. Proc. Natl. Acad. Sci. USA 103, 14331–14336 (2006).

    Article  CAS  Google Scholar 

  28. Frey, P.A. Radical mechanisms of enzymatic catalysis. Annu. Rev. Biochem. 70, 121–148 (2001).

    Article  CAS  Google Scholar 

  29. Berkovitch, F., Nicolet, Y., Wan, J.T., Jarrett, J.T. & Drennan, C.L. Crystal structure of biotin synthase, an S-adenosylmethionine-dependent radical enzyme. Science 303, 76–79 (2004).

    Article  CAS  Google Scholar 

  30. Matthews, B.W. Solvent content of protein crystals. J. Mol. Biol. 33, 491–497 (1968).

    Article  CAS  Google Scholar 

  31. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  32. Schneider, T.R. & Sheldrick, G.M. Substructure solution with SHELXD. Acta Crystallogr. D Biol. Crystallogr. 58, 1772–1779 (2002).

    Article  Google Scholar 

  33. Uson, I. & Sheldrick, G.M. Advances in direct methods for protein crystallography. Curr. Opin. Struct. Biol. 9, 643–648 (1999).

    Article  CAS  Google Scholar 

  34. Otwinowski, Z. in CCP4 Proceedings (eds. Wolf, W., Evans, P.R. & Leslie, A.G.W.) 80–88 (SERC Daresbury Laboratory, Warrington, UK, 1991).

    Google Scholar 

  35. Terwilliger, T.C. Maximum-likelihood density modification. Acta Crystallogr. D Biol. Crystallogr. 56, 965–972 (2000).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  Google Scholar 

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

    Article  Google Scholar 

  39. Laskowski, R.A., Moss, D.S. & Thornton, J.M. Main-chain bond lengths and bond angles in protein structures. J. Mol. Biol. 231, 1049–1067 (1993).

    Article  CAS  Google Scholar 

  40. Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D Biol. Crystallogr. 60, 2256–2268 (2004).

    Article  CAS  Google Scholar 

  41. Kabsch, W., Kabsch, H. & Eisenberg, D. Packing in a new crystalline form of glutamine synthetase from Escherichia coli. J. Mol. Biol. 100, 283–291 (1976).

    Article  CAS  Google Scholar 

  42. Gouet, P., Courcelle, E., Stuart, D.I. & Metoz, F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305–308 (1999).

    Article  CAS  Google Scholar 

  43. Jones, S. & Thornton, J.M. Protein-protein interactions: a review of protein dimer structures. Prog. Biophys. Mol. Biol. 63, 31–65 (1995).

    Article  CAS  Google Scholar 

  44. Jones, S. & Thornton, J.M. Principles of protein-protein interactions. Proc. Natl. Acad. Sci. USA 93, 13–20 (1996).

    Article  CAS  Google Scholar 

  45. DeLano, W.L. The PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, California, USA, 2002).

    Google Scholar 

  46. Potterton, E., McNicholas, S., Krissinel, E., Cowtan, K. & Noble, M. The CCP4 molecular-graphics project. Acta Crystallogr. D Biol. Crystallogr. 58, 1955–1957 (2002).

    Article  Google Scholar 

  47. Martinez-Gomez, N.C. & Downs, D.M. ThiC is an [Fe-S] cluster protein that requires AdoMet to generate the 4-amino-5-hydroxymethyl-2-methylpyrimidine moiety in thiamin synthesis. Biochemistry 47, 9054–9056 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank NE-CAT beamline 24-ID-C, supported by US National Institutes of Health grant RR15301, for the use of beam time. We thank C. Kinsland (Cornell University Protein Facility) for the preparation of the HMP-P synthase overexpression plasmid and L. Kinsland for assistance in the preparation of this manuscript. This work was supported by US National Institutes of Health grants DK44083 (T.P.B.), GM63847 (S.J.B.) and DK67081 (S.E.E.), the Beckman Foundation (Young Investigator Award to C.K.) and the Dreyfus Foundation (Camille Dreyfus Teacher Scholar Award to C.K.). S.E.E. is indebted to the W.M. Keck Foundation and the Lucille P. Markey Charitable Trust.

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Author notes

  1. Abhishek Chatterjee and Yue Li: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, 120 Baker Laboratory, Cornell University, Ithaca, 14853-1301, New York, USA

    Abhishek Chatterjee, Yue Li, Yang Zhang, Tadhg P Begley & Steven E Ealick

  2. Department of Chemistry, 104 Chemistry Building, The Pennsylvania State University, University Park, 16802, Pennsylvania, USA

    Tyler L Grove, Carsten Krebs & Squire J Booker

  3. Department of Biochemistry and Molecular Biology, 108 Althouse Laboratory, The Pennsylvania State University, University Park, 16802, Pennsylvania, USA

    Michael Lee, Carsten Krebs & Squire J Booker

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  1. Abhishek Chatterjee
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Contributions

A.C. performed all biochemical studies. Y.L. and Y.Z. performed all crystallographic studies. T.L.G. assisted in protein purification and EPR spectroscopy. M.L. recorded and analyzed Mössbauer spectra. C.K. and S.J.B. directed the EPR and Mössbauer studies, T.P.B. directed the biochemical studies and S.E.E. directed the crystallographic studies.

Corresponding authors

Correspondence to Tadhg P Begley or Steven E Ealick.

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Chatterjee, A., Li, Y., Zhang, Y. et al. Reconstitution of ThiC in thiamine pyrimidine biosynthesis expands the radical SAM superfamily. Nat Chem Biol 4, 758–765 (2008). https://doi.org/10.1038/nchembio.121

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