Molecular Cloning and Expression of a Mouse Thiamin Pyrophosphokinase cDNA

Nosaka, Kazuto; Onozuka, Mari; Nishino, Hoyoku; Nishimura, Hiroshi; Kawasaki, Yuko; Ueyama, Hisao

doi:10.1074/jbc.274.48.34129

Cited by 25 publications

(11 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It should be noted, however, that a low level of expression does not necessarily mean that the protein has little biological importance. For instance, low amounts of mRNA were reported for mouse brain thiamine pyrophosphokinase (19), yet this enzyme is absolutely required for ThDP synthesis and hence for brain oxidative metabolism. In the case of ThTPase, the fact that it is a relatively rare protein in most tissues is compensated, at least to some extent, by its relatively high catalytic efficiency.…”

Section: Resultsmentioning

confidence: 99%

“…Both were cloned by reverse transcription-PCR, the human enzyme was functionally expressed in Escherichia coli, and its distribution in human tissue was investigated. After the high affinity thiamine transporter, whose mutation causes thiamine-responsive megaloblastic anaemia (18), and thiamine pyrophosphokinase (19), ThTPase is the third protein of thiamine metabolism to be characterized in mammals.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Molecular Characterization of a Specific Thiamine Triphosphatase Widely Expressed in Mammalian Tissues

Lakaye¹,

Makarchikov²,

Antunes³

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues, and recent data suggest that it may act as a phosphate donor for the phosphorylation of some proteins. In the mammalian brain, ThTP synthesis is rapid, but its steady-state concentration remains low, presumably because of rapid hydrolysis. In this report we purified a soluble thiamine triphosphatase (ThTPase; EC 3.6.1.28) from calf brain. The bovine ThTPase is a 24-kDa monomer, hydrolyzing ThTP with virtually absolute specificity. Partial sequence data obtained from the purified bovine enzyme by tandem mass spectrometry were used to search the GenBank TM data base. A significant identity was found with only one human sequence, the hypothetical 230-amino acid protein MGC2652. The coding regions from human and bovine brain mRNA were amplified by reverse transcription-PCR, cloned in Escherichia coli, and sequenced. The human open reading frame was expressed in E. coli as a GST fusion protein. Transformed bacteria had a high isopropyl-␤-D-thiogalactopyranoside-inducible ThTPase activity. The recombinant ThTPase had properties similar to those of human brain ThTPase, and it was specific for ThTP. The mRNA was expressed in most human tissues but at relatively low levels. This is the first report of a molecular characterization of a specific ThTPase.

show abstract

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Molecular Characterization of a Specific Thiamine Triphosphatase Widely Expressed in Mammalian Tissues

Lakaye¹,

Makarchikov²,

Antunes³

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The amino acid sequence of TPK was first determined in Saccharomyces cerevisiae (Nosaka et al 1993) and its crystal structure has been resolved . Today the TPK mechanism is well understood, its activity has been reported in both higher eukaryotes (Mitsuda et al 1979;Nosaka et al 1999Nosaka et al , 2001Wakabayashi et al 1979) and prokaryotes, and the crystal structure of the mammalian TPK is also available .…”

Section: Introductionmentioning

confidence: 99%

Thiamin pyrophosphokinase is required for thiamin cofactor activation in Arabidopsis

et al. 2007

View full text Add to dashboard Cite

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.

show abstract

“…32 P-labeled TPP was generated catalytically from [␥-32 P]ATP and thiamine by using thiamine pyrophosphokinase as described in ref. 23. ITC was performed by using a VP-ITC (Microcal).…”

Section: Methodsmentioning

confidence: 99%

Thiamine biosynthesis in algae is regulated by riboswitches

Croft

Moulin

Webb

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

215

197

View full text Add to dashboard Cite

In bacteria, many genes involved in the biosynthesis of cofactors such as thiamine pyrophosphate (TPP) are regulated by riboswitches, regions in the 5 end of mRNAs to which the cofactor binds, thereby affecting translation and/or transcription. TPP riboswitches have now been identified in fungi, in which they alter mRNA splicing. Here, we show that addition of thiamine to cultures of the model green alga Chlamydomonas reinhardtii alters splicing of transcripts for the THI4 and THIC genes, encoding the first enzymes of the thiazole and pyrimidine branches of thiamine biosynthesis, respectively, concomitant with an increase in intracellular thiamine and TPP levels. Comparison with Volvox carteri, a related alga, revealed highly conserved regions within introns of these genes. Inspection of the sequences identified TPP riboswitch motifs, and RNA transcribed from the regions binds TPP in vitro. The THI4 riboswitch, but not the promoter region, was found to be necessary and sufficient for thiamine to repress expression of a luciferase-encoding reporter construct in vivo. The pyr1 mutant of C. reinhardtii, which is resistant to the thiamine analogue pyrithiamine, has a mutation in the THI4 riboswitch that prevents the THI4 gene from being repressed by TPP. By the use of these riboswitches, thiamine biosynthesis in C. reinhardtii can be effectively regulated at physiological concentrations of the vitamin.regulation of metabolism ͉ Chlamydomonas reinhardtii ͉ luciferase reporter gene ͉ alternative splicing of mRNA T hiamine (vitamin B 1 ) is synthesized via a branched pathway ( Fig. 1) from the condensation of a thiazole and a pyrimidine moiety to make thiamine monophosphate (TMP). This is then phosphorylated to make thiamine pyrophosphate (TPP), the active cofactor, either directly in prokaryotes, or by dephosphorylation followed by pyrophosphorylation in eukaryotes. Like many cofactors, the level of TPP within the cell is low, the majority of it being associated with its cognate enzymes, which are part of the central respiratory pathways. The biosynthetic pathway is carefully regulated to ensure that TPP production meets cellular demand, usually by regulation of gene expression for one or more of the enzymes, and in bacteria and fungi exogenous thiamine severely represses gene expression (1, 2).In bacteria, an important TPP regulatory mechanism is mediated via riboswitches in one or more of the thiamine biosynthesis genes. Riboswitches are short sequences in mRNAs that bind metabolites directly, without the need for intermediary proteins. Binding of the ligand alters the secondary structure of the RNA, thereby regulating expression of the gene, typically by premature transcription termination and/or initiation of translation (3, 4). Until recently, most of the work on riboswitches was carried out in prokaryotic systems. Now, TPP riboswitches have been found in the 5Ј UTR of genes for thiamine biosynthetic enzymes in the fungi Aspergillus oryzae and Neurospora crassa, where they appear to operate by causing alternative...

show abstract

Molecular Cloning and Expression of a Mouse Thiamin Pyrophosphokinase cDNA

Cited by 25 publications

References 27 publications

Molecular Characterization of a Specific Thiamine Triphosphatase Widely Expressed in Mammalian Tissues

Molecular Characterization of a Specific Thiamine Triphosphatase Widely Expressed in Mammalian Tissues

Thiamin pyrophosphokinase is required for thiamin cofactor activation in Arabidopsis

Thiamine biosynthesis in algae is regulated by riboswitches

Contact Info

Product

Resources

About