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Molecular characterization of thymidine kinase from Ureaplasma urealyticum : nucleoside analogues as potent inhibitors of mycoplasma growth
Author(s) -
Carnrot Cecilia,
Wehelie Rahma,
Eriksson Staffan,
Bölske Göran,
Wang Liya
Publication year - 2003
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2003.03717.x
Subject(s) - ureaplasma urealyticum , biology , thymidine kinase , thymidine , nucleoside , biochemistry , nucleotide salvage , mycoplasma , microbiology and biotechnology , virology , in vitro , nucleotide , gene , virus , herpes simplex virus
Summary Ureaplasma urealyticum ( U. urealyticum ), belonging to the class Mollicutes, is a human pathogen colonizing the urogenital tract and causes among other things respiratory diseases in premature infants. We have studied the salvage of pyrimidine deoxynucleosides in U. urealyticum and cloned a key salvage enzyme, thymidine kinase (TK) from U. urealyticum . Recombinant Uu ‐TK was expressed in E. coli , purified and characterized with regards to substrate specificity and feedback inhibition. Uu ‐TK efficiently phosphorylated thymidine (dThd) and deoxyuridine (dUrd) as well as a number of pyrimidine nucleoside analogues. All natural ribonucleoside/deoxyribonucleoside triphosphates, except dTTP, served as phosphate donors, while dTTP was a feedback inhibitor. The level of Uu ‐TK activity in U. urealyticum extracts increased upon addition of dUrd to the growth medium. Fluoropyrimidine nucleosides inhibited U. urealyticum and M. pneumoniae growth and this inhibitory effect could be reversed by addition of dThd, dUrd or deoxytetrahydrouridine to the growth medium. Thus, the mechanism of inhibition was most likely the depletion of dTTP, either via a blocked thymidine kinase reaction and/or thymidylate synthesis step and these metabolic reactions should be suitable targets for antimycoplasma chemotherapy.