A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis.
We report the characterization of a Brassica napus cDNA clone (pBTHI) encoding a protein (BTHI) 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.
Kim
Y S
YS
Department of Life Science and School of Environmental Engineering, Pohang University of Science and Technology, Kyungbuk, South Korea.
Nosaka
K
K
Downs
D M
DM
Kwak
J M
JM
Park
D
D
Chung
I K
IK
Nam
H G
HG
D019883:Alkyl and Aryl Transferases / Q000235:genetics*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics; D001937:Brassica / Q000201:enzymology / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000235:genetics*; D010940:Plant Proteins; D018749:RNA, Plant / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013831:Thiamine / Q000096:biosynthesis*
D018076:DNA, Complementary; D009097:Multienzyme Complexes; D010940:Plant Proteins; D018749:RNA, Plant; C113853:BTH1 protein, Brassica napus; D019883:Alkyl and Aryl Transferases; D017856:Phosphotransferases (Phosphate Group Acceptor); D013831:Thiamine