Open AccessCloning and characterization of the thiD/J gene of Escherichia coli encoding a thiamin-synthesizing bifunctional enzyme, hydroxymethylpyrimidine kinase/phosphomethylpyrimidine kinase
Author(s) -
Tomoko Mizote,
Masashi Tsuda,
D. G. Smith,
Hideo Nakayama,
Takahiro Nakazawa
Publication year - 1999
Publication title -
microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.019
H-Index - 179
eISSN - 1465-2080
pISSN - 1350-0872
DOI - 10.1099/13500872-145-2-495
Subject(s) - microbiology and biotechnology , map3k7 , biology , phosphofructokinase 2 , orfs , gene , biochemistry , open reading frame , kinase , protein kinase a , sequence analysis , peptide sequence , mitogen activated protein kinase kinase
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.