Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2188279 | Journal of Molecular Biology | 2007 | 10 Pages |
In antiviral and cancer therapy, deoxyribonucleoside kinases (dNKs) are often the rate-limiting step in activating nucleoside analog (NA) prodrugs into their cytotoxic, phosphorylated forms. We have constructed libraries of hybrid enzymes by non-homologous recombination of the pyrimidine-specific human thymidine kinase 2 and the broad-specificity dNK from Drosophila melanogaster; their low sequence identity has precluded engineering by conventional, homology-dependent shuffling techniques. From these libraries, we identified chimeras that phosphorylate nucleoside analogs with higher activity than either parental enzyme, and that possess new activity towards the anti-HIV prodrug 2′,3′-didehydro-3′-deoxythymidine (d4T). These results demonstrate the potential of non-homologous recombination within the dNK family for creating enzymes with new and improved activities towards nucleoside analogs. In addition, our results exposed a previously unknown role for the C-terminal regions of these dNKs in determining substrate selectivity.