Molecular characterization of thymidine kinase from Ureaplasma urealyticum: nucleoside analogues as potent inhibitors of mycoplasma growth

Abstract

SummaryUreaplasma 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.

Imaging bacterial infections with radiolabeled 1-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-iodouracil

“…Finally, the sensitivity of E. coli to FIAU, coupled with the uptake of FIAU by several other bacteria, suggests that TK substrates could be used not only for imaging bacteria but also for therapy. Indeed, it has been shown that nucleoside analogues are potent inhibitors of the growth of mycoplasma and mycobacterial strains (26,27). Recognition of the widespread presence and unique substrate specificities of bacterial TKs may therefore prove useful for the design of novel diagnostic as well as therapeutic modalities.…”

Structures of thymidine kinase 1 of human and mycoplasmic origin

“…21. For production of selenomethionine (SeMet)-substituted Uu-TK, the Uu-TK construct was transformed into the chemical competent, methionine auxotrophic E. coli strain B834(DE3) (Novagen).…”

“…The SelenoMet media was changed to contain SeMet before induction with 1 mM isopropyl ␤-D-galactopyranoside for 6 h at 25°C, and SeMet-substituted Uu-TK was purified essentially as described in ref. 21.…”

“…Recently, Ureaplasma urealyticum TK (Uu-TK) was cloned and expressed and characterized biochemically (21). U. urealyticum is a mycoplasma species with a known genome of Ϸ600 genes (22).…”

“…These bacteria colonize the urogenital tract and are associated with infertility, altered sperm motility, and pneumonia in neonates (23). Initial characterization of Uu-TK demonstrated that it has similar enzyme kinetic properties to human TK1 (hTK1) regarding the narrow specificity for deoxyuridine and dT and with dTTP serving as a feedback inhibitor (21). No genes for the de novo synthesis of deoxyribonucleotides have been found in the U. urealyticum genome.…”