Deoxyribonucleoside kinases in bacteria, plants and humans

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

Pasteurella multocida thymidine kinase 1 efficiently activates pyrimidine nucleoside analogs.

In the Pasteurella multocida genome only one putative deoxyribonucleoside kinase encoding gene, for thymidine kinase 1 (PmTK1), was identified. The PmTK1 gene was sub-cloned into Escherichia coli KY895 and it sensitized the host towards 2',2'-difluoro-deoxycytidine (gemcitabine, dFdC), 3'-azido-thymidine (AZT) and 5-fluoro-deoxyuridine (5F-dU). PmTK1 was over-expressed and purified with two different tags. Apparently, deoxyuridine (dU), and not thymidine (dT), is the preferred substrate. We suggest that PmTK1s could be employed as a species-specific activator of uracil-based nucleoside antibiotics

Plants salvage deoxyribonucleosides in mitochondria.

Deoxyribonucleoside kinases phosphorylate deoxyribonucleosides into the corresponding 5'-monophosphate deoxyribonucleosides to supply the cell with nucleic acid precursors. In mitochondrial fractions of the model plant Arabidopsis thaliana, we detected deoxyadenosine and thymidine kinase activities, while the cytosol fraction contained six-fold lower activity and chloroplasts contained no measurable activities. In addition, a mitochondrial fraction isolated from the potato Solanum tuberosum contained thymidine kinase and deoxyadenosine kinase activities. We conclude that an active salvage of deoxyribonucleosides in plants takes place in their mitochondria. In general, the observed localization of the plant dNK activities in the mitochondrion suggests that plants have a different organization of the deoxyribonucleoside salvage compared to mammals

Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana

Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (dNKs) salvage deoxyribonucleosides by transfer of a phosphate group to the 5' of a deoxyribonucleoside. This salvage pathway is well characterized in mammals but in contrast little is known about how plants salvage deoxyribonucleosides. We show that during salvage, deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding mono-phosphate compounds with a surprising preference for purines over pyrimidines. Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1-like genes (AtTK1a and AtTK1b) and deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T-DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, but AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a and AtTK1b catalyze redundant reactions. Our results point out a crucial role for salvage of thymidine during early plant development. © 2012 The Authors Journal compilation © 2012 FEBS

Bacterial Deoxyribonucleoside Kinases Are Poor Suicide Genes in Mammalian Cells

Transfer of deoxyribonucleoside kinases (dNKs) into cancer cells increases the activity of cytotoxic nucleoside analogues. It has been shown that bacterial dNKs, when introduced into Escherichia coli, sensitize this bacterium toward nucleoside analogues. We studied the possibility of using bacterial dNKs, for example deoxyadenosine kinases (dAKs), to sensitize human cancer cells to gemcitabine. Stable and transient transfections of bacterial dNKs into human cells showed that these were much less active than human and fruitfly dNKs. The fusion of dAK from Bacillus cereus to the green fluorescent protein induced a modest sensitization. Apparently, bacterial dNKs did not get properly expressed or are unstable in the mammalian cell

Structural studies of thymidine kinases from Bacillus anthracis and Bacillus cereus provide insights into quaternary structure and conformational changes upon substrate binding

Thymidine kinase (TK) is the key enzyme in salvaging thymidine to produce thymidine monophosphate. Owing to its ability to phosphorylate nucleoside analogue prodrugs, TK has gained attention as a rate-limiting drug activator. We describe the structures of two bacterial TKs, one from the pathogen Bacillus anthracis in complex with the substrate dT, and the second from the food-poison-associated Bacillus cereus in complex with the feedback inhibitor dTTP. Interestingly, in contrast with previous structures of TK in complex with dTTP, in this study dTTP occupies the phosphate donor site and not the phosphate acceptor site. This results in several conformational changes compared with TK structures described previously. One of the differences is the way tetramers are formed. Unlike B. anthracis TK, B. cereus TK shows a loose tetramer. Moreover, the lasso-domain is in open conformation in B. cereus TK without any substrate in the active site, whereas in B. anthracis TK the loop conformation is closed and thymidine occupies the active site. Another conformational difference lies within a region of 20 residues that we refer to as phosphate-binding beta-hairpin. The phosphate-binding beta-hairpin seems to be a flexible region of the enzyme which becomes ordered upon formation of hydrogen bonds to the alpha-phosphate of the phosphate donor, dTTP. In addition to descriptions of the different conformations that TK may adopt during the course of reaction, the oligomeric state of the enzyme is investigated

Characterization of oligomeric and kinetic properties of tomato thymidine kinase 1.

The gene encoding thymidine kinase 1 from tomato (toTK1) has in combination with azidothymidine (AZT) recently been proposed as a powerful suicide gene for anticancer gene therapy. The toTK1/AZT combination has been demonstrated to have several advantages for the treatment of glioblastomas because AZT can easily penetrate the blood-brain barrier and toTK1 can efficiently phosphorylate AZT and also AZT-monophosphate. In a pursuit to further understand the properties of toTK1, we examined the oligomerization properties of recombinant toTK1 and its effect on enzyme kinetics. Previously, it has been shown that human TK1 is a dimer in the absence of ATP and a tetramer if preincubated with ATP. However, we show here that ATP preincubation did not result in a structural shift from dimer to tetramer in toTK1. For human TK1 pretreated with ATP, the K(m) value decreased 20-fold, but toTK1's K(m) value did not show a dependence on the presence or absence of ATP. Furthermore, toTK1 was always found in a highly active form

Drosophila melanogaster deoxyribonucleoside kinase activates gemcitabine

Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) can additionally sensitize human cancer cell lines towards the anti-cancer drug gemcitabine. We show that this property is based on the Dm-dNK ability to efficiently phosphorylate gemcitabine. The 2.2 angstrom resolution structure of DmdNK in complex with gemcitabine shows that the residues Tyr70 and Arg105 play a crucial role in the firm positioning of gemcitabine by extra interactions made by the fluoride atoms. This explains why gemcitabine is a good substrate for Dm-dNK(. (C) 2009 Elsevier Inc. All rights reserved