Expression of human mitochondrial thymidine kinase in escherichia coli: correlation between the enzymatic activity of pyrimidine nucleoside analogues and their inhibitory effect on bacterial growth

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Abstract

Mitochondrial thymidine kinase (TK2) phosphorylates pyrimidine nucleosides to monophosphates and is expressed constitutively through the cell cycle in all cells. Because of the overlap of its substrate specificity with that of the cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK), it has been difficult to determine the role of TK2 in activating nucleosides used in chemotherapy. In this report, we described the construction of a recombinant Escherichia coli strain which could be used to test if TK2 activity is limiting for the toxicity of nucleosides. Enzymes of bacterial origin which are involved in thymidine and deoxyuridine anabolism and catabolism were eliminated, and the cDNA for human TK2 was introduced. In the crude extract of the engineered E. coli, the level of thymidine kinase was, after induction of TK2 expression, several hundred fold higher than in the control strain. Several pharmacologically interesting nucleoside analogues, including 3′-azidothymidine, 2′,3′-didehydro-2′,3′-dideoxythymidine, and 2′,3′-dideoxy-β-l-3′-thiacytidine, were tested for their effects on the growth of this recombinant strain. For a comparison, the phosphorylation of these compounds was determined with purified recombinant TK1, TK2, and dCK. A correlation was observed between the phosphorylation of several of these compounds by TK2 and their effects on bacterial growth. These results demonstrate that activation of growth-inhibiting pyrimidine nucleosides can be catalyzed by TK2, and together with recombinant E. coli strains expressing other cellular nucleoside kinases, this whole-cell bacterial system may serve as a tool to predict the efficacy and side effects of chemotherapeutic nucleosides.

Section snippets

Bacterial strains and growth media

The bacterial strains used in this study were all derivatives of E. coli K12 and are listed in Table 1. As rich medium, Luria broth (LB) was used [8]. The minimal medium was AB medium [9] supplemented with 0.2% glucose, 0.2% vitamin-free casamino acids, and 1 μg/mL thiamine. When required, tryptophan was added at 50 μg/mL and uridine at 20 μg/mL (82 μM). Antibiotics were used in the following final concentrations: ampicillin, 100 μg/mL; tetracycline, 10 μg/mL; and kanamycin, 30 μg/mL.

Nucleoside analogues

Most

Activity of TK and dCK in crude extracts from the recombinant bacteria

To ascertain the expression of human TK2 in the engineered E. coli, crude extracts from strain SØ5338, containing the plasmid-borne coding sequence of human TK2 and the control strain SØ5292 (Table 1), were assayed for TK and dCK activity (Table 2). Both strains were devoid of endogenous E. coli TK activity due to the tdk-1 mutation. Since TK2 overlaps in its substrate specificity with dCK, the TK and dCK activities of strain SØ5218 (Table 1), harboring the coding sequence of human dCK on a

Discussion

5-Fluorouracil was designed for use as an antineoplastic agent [14], and 5FdU is one of its metabolites in vivo[15]. In cells, 5FdU has several metabolic routes. It is mainly phosphorylated by TK1 and TK2 to form 5FdU monophosphate, which can bind tightly to thymidylate synthase and inhibit the enzyme, thus impeding the transformation of dUMP to dTMP 15, 16. 5FdU can also be converted to 5-fluorouracil through a reversible reaction catalyzed by thymidine phosphorylase or, less efficiently,

Acknowledgements

This work was funded by the Swedish Medical Research Council, Swedish National Science Research Council, and grants from the EU Commission (BMH4-CT96-0479, to S.E.) and the Danish National Research Foundation (to J.N.).

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