A BRASSICA CDNA CLONE ENCODING A BIFUNCTIONAL HYDROXYMETHYLPYRIMIDINEKINASE THIAMIN-PHOSPHATE PYROPHOSPHORYLASE INVOLVED IN THIAMIN BIOSYNTHESIS/

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 yl-4-amino-5-hydroxymethylpyrimidine-monophosphate kinase (HMP-P kinase). The cDNA clone was isolated by a novel functional complementa tion strategy employing an Escherichia coli mutant deficient in the TM P-PPase activity. A biochemical assay showed the clone to confer recov ery 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% se quence similarity to the N-terminal TMP-PPase region of the bifunction al yeast proteins Saccharomyces THI6 and Schizosaccharomyces pombe THI 4, respectively. The N-terminal part of BTH1 showed 58% sequence simil arity to HMP-P kinase of Salmonella typhimurium. The cDNA clone functi onally complemented the S. typhimurium and E. coli thiD mutants defici ent in the HMP-P kinase activity. These results show that the clone en codes 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 bifunctio nal proteins that encode TMP-PPase at the N-terminus and 4-methyl-5-(2 -hydroxyethyl)thiazole kinase at the C-terminus, Expression of the BTH 1 gene is negatively regulated by thiamin, as in the cases for the thi amin biosynthetic genes of microorganisms. This is the first report of a plant thiamin biosynthetic gene on which a specific biochemical act ivity is assigned. The Brassica BTH1 gene may correspond to the Arabid opsis TH-1 gene.