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Plant Molecular Biology

, Volume 65, Issue 1–2, pp 151–162 | Cite as

Thiamin pyrophosphokinase is required for thiamin cofactor activation in Arabidopsis

  • Imad Ajjawi
  • Miguel A. Rodriguez Milla
  • John Cushman
  • David K. Shintani
Article

Abstract

Thiamin pyrophosphate (TPP) is an essential enzyme cofactor required for the viability of all organisms. Whether derived from exogenous sources or through de novo synthesis, thiamin must be pyrophosphorylated for cofactor activation. The enzyme thiamin pyrophosphokinase (TPK) catalyzes the conversion of free thiamin to TPP in plants and other eukaryotic organisms and is central to thiamin cofactor activation. While TPK activity has been observed in a number of plant species, the corresponding gene/protein has until now not been identified or characterized for its role in thiamin metabolism. Here we report the functional identification of two Arabidopsis TPK genes, AtTPK1 and AtTPK2 and the enzymatic characterization of the corresponding proteins. AtTPK1 and AtTPK2 are biochemically redundant cytosolic proteins that are similarly expressed throughout different plant tissues. The essential nature of TPKs in plant metabolism is reflected in the observation that while single gene knockouts of either AtTPK1 or AtTPK2 were viable, the double mutant possessed a seedling lethal phenotype. HPLC analysis revealed the double mutant is nearly devoid of TPP and instead accumulates the precursor of the TPK reaction, free thiamin. These results suggest that TPK activity provides the sole mechanism by which exogenous and de novo derived thiamin is converted to the enzyme cofactor TPP.

Keywords

Arabidopsisthaliana Cofactor activation Thiamin Thiamin pyrophosphate Thiamin pyrophosphokinase 

Abbreviations

TPK

Thiamin pyrophosphokinase

AtTPK1

Arabidopsis thaliana TPK locus At1g02880

AtTPK2

Arabidopsis thaliana TPK locus At2g44750

AtTPK1

Arabidopsis thaliana TPK protein 1 corresponding to the AtTPK1 locus

AtTPK2

Arabidopsis thaliana TPK protein 2 corresponding to the AtTPK2 locus

AtTPK1-KO

T-DNA insertion mutant line SALK_010916 corresponding to AtTPK1

AtTPK2-KO

T-DNA insertion mutant line SALK_011765 corresponding to AtTPK2

AtTPK1/2-KO

AtTPK1 and AtTPK2 double knockout

TMP

Thiamin monophosphate

TPP

Thiamin pyrophosphate

Notes

Acknowledgements

The authors would like to thank Dr Jen Sheen (Massachusetts General Hospital, Boston, MA) for providing p35s-sGFP-TYG-NOS and Dr Maureen Hanson (Cornell University, Ithaca, NY) for providing p35s35sAMV-coxIV-S65TmGFP4-NOS. We would also like to thank Katrina Meeth for technical support. We would also like to thank Drs Christie Howard, Jeff Harper and the members of the Shintani Lab for their constructive criticism. This work was supported by the National Science Foundation grant no. MCB-0236210.

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Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Imad Ajjawi
    • 1
  • Miguel A. Rodriguez Milla
    • 1
    • 2
  • John Cushman
    • 1
  • David K. Shintani
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of NevadaRenoUSA
  2. 2.Departamento de BiotecnologíaInstituto Nacional de Investigación y Tecnología Agraría y AlimentariaMadridSpain

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