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Saccharomyces cerevisiae THI4p is a suicide thiamine thiazole synthase

Nature volume 478, pages 542–546 (2011)Cite this article

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Abstract

Thiamine pyrophosphate 1 is an essential cofactor in all living systems1. Its biosynthesis involves the separate syntheses of the pyrimidine 2 and thiazole 3 precursors, which are then coupled2. Two biosynthetic routes to the thiamine thiazole have been identified. In prokaryotes, five enzymes act on three substrates to produce the thiazole via a complex oxidative condensation reaction, the mechanistic details of which are now well established2,3,4,5,6. In contrast, only one gene product is involved in thiazole biosynthesis in eukaryotes (THI4p in Saccharomyces cerevisiae)7. Here we report the preparation of fully active recombinant wild-type THI4p, the identification of an iron-dependent sulphide transfer reaction from a conserved cysteine residue of the protein to a reaction intermediate and the demonstration that THI4p is a suicide enzyme undergoing only a single turnover.

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Figure 1: Thiamine pyrophosphate and thiamine thiazole biosynthesis.
Figure 2: Identification of the site of the M − 34 Da modification in wtTHI4p.
Figure 3: Reconstitution of the biosynthesis of ADT 5.
Figure 4: Characterization of native THI4p from S. cerevisiae.

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Protein Data Bank

Data deposits

Atomic coordinates for the crystal structure of THI4p reported here have been deposited in the Protein Data Bank under accession code 3FPZ. This entry includes structure factors originally contained within PDB accession code 2GJC.

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Acknowledgements

We thank F. W. McLafferty for initial FTMS analysis of THI4p. This research was funded by NIH grant DK44083 (to T.P.B.), the Robert E. Welch Foundation grant A-0034 (to T.P.B.), DK67081 (to S.E.E.) and NSF grant DBI0821700 (to D.H.R.).

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Author notes

  1. Abhishek Chatterjee & Shridhar Bale

    Present address: Present address: The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA.,

Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Cornell University, Ithaca, 14853, New York, USA

    Abhishek Chatterjee, Shridhar Bale & Steven E. Ealick

  2. Department of Chemistry, Texas A&M University, College Station, Texas 77843, USA,

    N. Dinuka Abeydeera, Pei-Jing Pai, David H. Russell & Tadhg P. Begley

  3. Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California 92093, USA,

    Pieter C. Dorrestein

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Contributions

A.C. performed all biochemical experiments with recombinantly expressed THI4p, N.D.A. performed all biochemical experiments with endogenous THI4p from yeast, S.B. performed structural analyses, P.C.D. supervised FTMS experiments and assisted in FTMS data analyses, and P.-J.P. performed the sequence analysis by mass spectrometry on endogenous THI4p. D.H.R. supervised the mass spectrometric analysis on endogenous THI4p, S.E.E. supervised the structural studies and T.P.B. supervised the biochemical studies.

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Correspondence to Steven E. Ealick or Tadhg P. Begley.

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The authors declare no competing financial interests.

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Chatterjee, A., Abeydeera, N., Bale, S. et al. Saccharomyces cerevisiae THI4p is a suicide thiamine thiazole synthase. Nature 478, 542–546 (2011). https://doi.org/10.1038/nature10503

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