Expression of human mitochondrial thymidine kinase in Escherichia coli: Correlation between the enzymatic activity of pyrimidine nucleoside analoguesand their inhibitory effect on bacterial growth

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 o f the cytosolic thymidine kinase (TiK1) and deoxycytidine kinase (dCK), it has been difficult to determine the role of TK2 in activating nucleosides u sed in chemotherapy. In this report, we described the construction of a rec ombinant Escherichia coli strain which could be used to test if TK2 activit y is limiting for the toxicity of nucleosides. Enzymes of bacterial origin which are involved in thymidine and deoxyuridine anabolism and catabolism w ere eliminated, and the cDNA for human TK2 was introduced. In the crude ext ract of the engineered E. coli, the level of thymidine kinase was, after in duction of TK2 expression, several hundred fold higher than in the control strain. Several pharmacologically interesting nucleoside analogues, includi ng 3'-azidothymidine, 2',3'-didehydro-2',3'-dideoxythymidine, and 2',3'-did eoxy-beta-L-3'-thiacytidine, were tested for their effects on the growth of this recombinant strain. For a comparison, the phosphorylation of these co mpounds was det ermined with purified recombinant TK1, TK2, and dCK. A corr elation was observed between the phosphorylation of several of these compou nds by TK2 and their effects on bacterial growth. These results demonstrate that activation of growth-inhibiting pyrimidine nucleosides can be catalyz ed by TK2, and together with recombinant E. coli strains expressing other c ellular nucleoside kinases, this whole cell bacterial system may serve as a tool to predict the efficacy and side effects of chemotherapeutic nucleosi des. (C) 2000 Elsevier Science Inc.