Deoxyribonucleoside kinases in bacteria, plants and humans

Abstract: Deoxyribonucleoside triphosphates (dNTPs) are the building blocks of DNA and can be synthesized either de novo or via salvage pathways. The first reaction in the salvage pathway of dNTPs is the conversion of deoxyribonucleosides (dN) into the deoxyribonucleoside monophosphate (dNMP). The corresponding enzymes, deoxyribonucleoside kinases (dNKs), are also important for the activation of many nucleoside analogs used in anti-viral and anti-cancer therapy. Nucleoside analogs mimic the natural DNA precursors and can interfere, as antimetabolites, with DNA synthesis leading to cell-death. In this thesis I studied the diversity of deoxyribonucleoside kinases in Gram-negative and Gram-positive bacteria. Several of the microbial deoxyribonucleoside kinases were over-expressed, purified and characterized for their substrate specificity, kinetics and structure function relationship. Nucleoside analogs were studied for their antibacterial potential and we show in this study that microbial deoxyribonucleoside kinases could activate nucleoside analogs, like 3’-azido-thymidine (AZT) in Gram-negative bacteria, and 2’,2’-difluorodeoxycytidine in Gram-positive bacteria. Human thymidine kinase 1 (TK1) has a very strict substrate specificity, and via a random mutagenesis approach I identified mutants with an increased selectivity towards nucleoside analogs, like AZT and 5-ethyl-deoxyuridine. I show that a decrease of kcat for thymidine is the main reason that TK1 mutants sensitize the host against analogs. Several animal and bacterial deoxyribonucleoside kinases have so far been thoroughly investigated, but the salvage of deoxyribonucleosides in plants is so far largely unknown. Here, I show that plants salvage deoxyribonucleosides mainly in the mitochondria and our plant model Arabidopsis thaliana contains two TK1s and an ancestor-like multisubstrate dNK. I also show that the TK1 activity is essential during the first stages of plant development.