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

    Research output: Contribution to journalArticlepeer-review

    39 Citations (Scopus)

    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 thiD/J.

    Original languageEnglish
    Pages (from-to)495-501
    Number of pages7
    JournalMicrobiology
    Volume145
    Issue number2
    DOIs
    Publication statusPublished - 1999 Feb

    Keywords

    • Bifunctional enzyme
    • Escherichia coli
    • Hydroxymethylpyrimidine kinase
    • Thiamin biosynthesis

    ASJC Scopus subject areas

    • Microbiology

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