Cloning and characterization of the thiD/J gene of Escherichia coli encoding a thiamin-synthesizing bifunctional enzyme, hydroxymethylpyrimidine kinase/phosphomethylpyrimidine kinase

Tomoko Mizote; Masataka Tsuda; D D S Smith; Hideo Nakayama; Teruko Nakazawa

doi:10.1099/13500872-145-2-495

Cloning and characterization of the thiD/J gene of Escherichia coli encoding a thiamin-synthesizing bifunctional enzyme, hydroxymethylpyrimidine kinase/phosphomethylpyrimidine kinase

Microbiology (Reading). 1999 Feb:145 ( Pt 2):495-501. doi: 10.1099/13500872-145-2-495.

Authors

Tomoko Mizote^{1

2}, Masataka Tsuda³, D D S Smith⁴, Hideo Nakayama², Teruko Nakazawa¹

Affiliations

¹ Department of Microbiology, Yamaguchi University School of Medicine, Ube 755-8505, Japan.
² Department of Food and Nutrition, Yamaguchi Prefectural University, Sakurabatake, Yamaguchi 753-8502, Japan.
³ Institute of Genetic Ecology, Tohoku University, Katahira, Sendai 980-8577, Japan.
⁴ Department of Food Macromolecular Science, Institute of Food Research, Earley Gate, Whiteknights Road, Reading RG6 6BZ, UK.

PMID: 10075431
DOI: 10.1099/13500872-145-2-495

Abstract

A 1.7 kb DNA fragment isolated from an E. coli genomic library was able to complement the thiamin requirement of strains carrying the thiM, thiJ and thiD mutations. The three genes encode hydroxyethylthiazole kinase, hydroxymethylpyrimidine (HMP) kinase and phosphomethylpyrimidine (HMP-p) kinase, respectively. Sequence analysis revealed that the 1.7 kb fragment contained two ORFs of 708 bp and 801 bp. The former ORF complemented the thiM mutation and the latter ORF both the thiJ and thiD mutations. The latter ORF was cloned into the expression vector pET3a, and the encoded protein was purified through three successive column chromatographies. The purified protein was able to convert HMP to its monophosphate and the monophosphate to its pyrophosphate. These results suggest that the two distinct enzyme activities, HMP kinase and HMP-P kinase, are indeed a bifunctional enzyme encoded by a single gene, designated thiDIJ.

MeSH terms

Amino Acid Sequence
Base Sequence
Cloning, Molecular
Escherichia coli / enzymology*
Escherichia coli / genetics
Genes, Bacterial
Molecular Sequence Data
Multienzyme Complexes / chemistry
Multienzyme Complexes / genetics*
Multienzyme Complexes / isolation & purification
Multienzyme Complexes / metabolism
Phosphotransferases (Alcohol Group Acceptor) / chemistry
Phosphotransferases (Alcohol Group Acceptor) / genetics*
Phosphotransferases (Alcohol Group Acceptor) / isolation & purification
Phosphotransferases (Alcohol Group Acceptor) / metabolism
Phosphotransferases (Phosphate Group Acceptor) / chemistry
Phosphotransferases (Phosphate Group Acceptor) / genetics*
Phosphotransferases (Phosphate Group Acceptor) / isolation & purification
Phosphotransferases (Phosphate Group Acceptor) / metabolism
Plasmids / genetics
Restriction Mapping
Sequence Analysis, DNA
Thiamine / biosynthesis

Substances

Multienzyme Complexes
Phosphotransferases (Alcohol Group Acceptor)
hydroxymethylpyrimidine kinase
Phosphotransferases (Phosphate Group Acceptor)
phosphomethylpyrimidine kinase
Thiamine

Associated data

GENBANK/D84200
GENBANK/D88442