Plant Molecular Biology

, Volume 37, Issue 6, pp 955–966 | Cite as

A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis

  • Yang Suk Kim
  • Kazuto Nosaka
  • Diana M. Downs
  • June Myoung Kwak
  • Deokhoon Park
  • II Kyung Chung
  • Hong Gil Nam
Article

Abstract

We report the characterization of a Brassica napus cDNA clone (pBTH1) encoding a protein (BTH1) with two enzymatic activities in the thiamin biosynthetic pathway, thiamin-phosphate pyrophosphorylase (TMP-PPase) and 2-methyl-4-amino-5-hydroxymethylpyrimidine-monophosphate kinase (HMP-P kinase). The cDNA clone was isolated by a novel functional complementation strategy employing an Escherichia coli mutant deficient in the TMP-PPase activity. A biochemical assay showed the clone to confer recovery of TMP-PPase activity in the E. coli mutant strain. The cDNA clone is 1746 bp long and contains an open reading frame encoding a peptide of 524 amino acids. The C-terminal part of BTH1 showed 53% and 59% sequence similarity to the N-terminal TMP-PPase region of the bifunctional yeast proteins Saccharomyces THI6 and Schizosaccharomyces pombe THI4, respectively. The N-terminal part of BTH1 showed 58% sequence similarity to HMP-P kinase of Salmonella typhimurium. The cDNA clone functionally complemented the S. typhimurium and E. coli thiD mutants deficient in the HMP-P kinase activity. These results show that the clone encodes a bifunctional protein with TMP-PPase at the C-terminus and HMP-P kinase at the N-terminus. This is in contrast to the yeast bifunctional proteins that encode TMP-PPase at the N-terminus and 4-methyl-5-(2-hydroxyethyl)thiazole kinase at the C-terminus. Expression of the BTH1 gene is negatively regulated by thiamin, as in the cases for the thiamin biosynthetic genes of microorganisms. This is the first report of a plant thiamin biosynthetic gene on which a specific biochemical activity is assigned. The Brassica BTH1 gene may correspond to the Arabidopsis TH-1 gene.

bifunctional enzyme Brassica napus cDNA hydroxymethylpyrimidine phosphate kinase thiamin thiamin phosphate pyrophosphorylase 

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Copyright information

© Kluwer Academic Publishers 1998

Authors and Affiliations

  • Yang Suk Kim
    • 1
  • Kazuto Nosaka
    • 2
  • Diana M. Downs
    • 3
  • June Myoung Kwak
    • 1
  • Deokhoon Park
    • 1
  • II Kyung Chung
    • 4
  • Hong Gil Nam
    • 1
  1. 1.Department of Life Science and School of Environmental EngineeringPohang University of Science and TechnologyPohang, KyungbukSouth Korea
  2. 2.Department of BiochemistryKyoto Prefectural University of MedicineKamigyo-ku, KyotoJapan
  3. 3.Department of BacteriologyUniversity of Wisconsin-MadisonMadisonUSA
  4. 4.Department of Horticultural ScienceCatholic University of Taegu-Hyosung, Hayang-upKyungsan, KyungbukSouth Korea

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