Non-homologous recombination of deoxyribonucleoside kinases from human and Drosophila melanogaster yields human-like enzymes with novel activities.
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.
Gerth
Monica L
ML
Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA 30322, USA.
Lutz
Stefan
S
D000818:Animals; D030561:Databases, Nucleic Acid; D003853:Deoxyribonucleosides / Q000737:chemistry / Q000378:metabolism; D004331:Drosophila melanogaster / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics*; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013937:Thymidine Kinase / Q000378:metabolism
D003853:Deoxyribonucleosides; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases; D013937:Thymidine Kinase