Plant Molecular Biology 37: 955–966, 1998.
© 1998 Kluwer Academic Publishers. Printed in Belgium.
A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine
kinase/thiamin-phosphate pyrophosphorylase involved in thiamin
Yang Suk Kim
, Kazuto Nosaka
, Diana M. Downs
, June Myoung Kwak
, Deokhoon Park
II Kyung Chung
and Hong Gil Nam
Department of Life Science and School of Environmental Engineering, Pohang University of Science and Technol-
ogy, Pohang, Kyungbuk, 790-784, South Korea (
author for correspondence);
Department of Biochemistry, Kyoto
Prefectural University of Medicine, Kamigyo-ku, Kyoto 602, Japan;
Department of Bacteriology, University of
Wisconsin-Madison, Madison, WI 53706, USA;
Department of Horticultural Science, Catholic University of
Taegu-Hyosung, Hayang-up, Kyungsan, Kyungbuk, 713-702, South Korea
Received 5 August 1997; accepted in revised form 25 February 1998
Key words: bifunctional enzyme, Brassica napus, cDNA, hydroxymethylpyrimidine phosphate kinase, thiamin,
thiamin phosphate pyrophosphorylase
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
regionof the bifunctional yeast proteinsSaccharomyces 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.
Thiamin (vitamin B-1), in the form of its diphosphate,
acts as a cofactor of many enzymes in key cellular
metabolic pathways such as citric acid cycle, glycoly-
sis, and pentose phosphate cycle [for a review, see 2].
The nucleotide sequence data reported will appear in the
GenBank and/EMBL Nucleotide Sequence Databases under the
accession number AF015310 (pBTH1 clone).
In man, thiamin is an essential nutrient. The thiamin
deﬁciency disease beriberi disturbs the central nervous
and circulatory systems due to accumulation of pyru-
vate and lactate. This disease has been a prevailing
health problem in rice-consuming countries, since rice
polishing removes most of thiamin in the bran .
Extensive genetic and biochemical studies in mi-
croorganisms such as Escherichia coli and yeasts
revealed some steps of the thiamin biosynthetic path-