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

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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.

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Authors and Affiliations

  1. Department of Life Science and School of Environmental Engineering, Pohang University of Science and Technology, Pohang, Kyungbuk, 790-784, South Korea

    Yang Suk Kim, June Myoung Kwak, Deokhoon Park & Hong Gil Nam

  2. Department of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto, 602, Japan

    Kazuto Nosaka

  3. Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, 53706, USA

    Diana M. Downs

  4. Department of Horticultural Science, Catholic University of Taegu-Hyosung, Hayang-up, Kyungsan, Kyungbuk, 713-702, South Korea

    II Kyung Chung

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  1. Yang Suk Kim
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Suk Kim, Y., Nosaka, K., Downs, D.M. et al. A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis. Plant Mol Biol 37, 955–966 (1998). https://doi.org/10.1023/A:1006030617502

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