Article

Plant Molecular Biology

, Volume 37, Issue 6, pp 955-966

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

  • Yang Suk KimAffiliated withDepartment of Life Science and School of Environmental Engineering, Pohang University of Science and Technology
  • , Kazuto NosakaAffiliated withDepartment of Biochemistry, Kyoto Prefectural University of Medicine
  • , Diana M. DownsAffiliated withDepartment of Bacteriology, University of Wisconsin-Madison
  • , June Myoung KwakAffiliated withDepartment of Life Science and School of Environmental Engineering, Pohang University of Science and Technology
  • , Deokhoon ParkAffiliated withDepartment of Life Science and School of Environmental Engineering, Pohang University of Science and Technology
  • , II Kyung ChungAffiliated withDepartment of Horticultural Science, Catholic University of Taegu-Hyosung, Hayang-up
  • , Hong Gil NamAffiliated withDepartment of Life Science and School of Environmental Engineering, Pohang University of Science and Technology

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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.

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