A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis
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- Title
- A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis
- Authors
- Kim, YS; Nosaka, K; Downs, DM; Kwak, JM; Park, D; Chung, IK; Nam, HG
- Date Issued
- 1998-08
- Publisher
- KLUWER ACADEMIC PUBL
- 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.
- Keywords
- bifunctional enzyme; Brassica napus; cDNA; hydroxymethylpyrimidine phosphate kinase; thiamin; thiamin phosphate pyrophosphorylase; REDUCTASE-THYMIDYLATE SYNTHASE; ARABIDOPSIS-THALIANA; ESCHERICHIA-COLI; SACCHAROMYCES-CEREVISIAE; FUNCTIONAL COMPLEMENTATION; HYDROXYETHYLTHIAZOLE KINASE; PISUM-SATIVUM; GENE; ALIGNMENT; SEQUENCES
- URI
- https://oasis.postech.ac.kr/handle/2014.oak/20704
- DOI
- 10.1023/A:1006030617502
- ISSN
- 0167-4412
- Article Type
- Article
- Citation
- PLANT MOLECULAR BIOLOGY, vol. 37, no. 6, page. 955 - 966, 1998-08
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