Nucleoside analogues (NA) are
prodrugs that are phosphorylated by
deoxyribonucleoside kinases (dNKs) as the first step towards a compound toxic to the cell. During the last 20 years, research around dNKs has gone into new organisms other than mammals and viruses. Newly discovered dNKs have been tested as
enzymes for suicide gene therapy. The tomato
thymidine kinase 1 (ToTK1) is a dNK that has been selected for its in vitro kinetic properties and then successfully been tested in vivo for the treatment of
malignant glioma. We present the selection of two improved variants of ToTK1 generated by random
protein engineering for suicide gene therapy with the NA
azidothymidine (AZT).We describe their selection, recombinant production and a subsequent kinetic and biochemical characterization. Their improved performance in killing of E. coli KY895 is accompanied by an increase in specificity for the NA AZT over the natural substrate
thymidine as well as a decrease in inhibition by
dTTP, the end product of the
nucleoside salvage pathway for
thymidine. The understanding of the enzymatic properties improving the variants efficacy is instrumental to further develop dNKs for use in suicide gene therapy.