Recombinant deoxyribonucleoside kinase from Drosophila melanogaster can improve gemcitabine based combined gene/chemotherapy for targeting cancer cells.

A recombinant deoxyribonucleoside kinase from Drosophila melanogaster with a deletion of the last 20 amino acid residues (named DmdNKΔC20) was hypothesized as a potential therapeutic tool for gene therapy due to its broad substrate specificity and better catalytic efficiency towards nucleosides and nucleoside analogs. This study was designed to evaluate the effect of DmdNKΔC20 for sensitizing human cancer cell lines to gemcitabine and to further investigate its role in reversal of acquired drug resistance in gemcitabine-resistant cancer cell line. The DmdNKΔC20 gene was delivered to three different cancer cell lines, including breast, colon and liver cancer cells, using lipid-mediated transfection reagent.

Gene duplications and losses among vertebrate deoxyribonucleoside kinases of the non-TK1 Family.

Deoxyribonucleoside kinases (dNKs) salvage deoxyribonucleosides (dNs) and catalyze the rate limiting step of this salvage pathway by converting dNs into corresponding monophosphate forms. These enzymes serve as an excellent model to study duplicated genes and their evolutionary history. So far, among vertebrates only four mammalian dNKs have been studied for their substrate specificity and kinetic properties.

Isolation of a novel protein, P12-from adult Drosophila melanogaster that inhibits deoxyribonucleoside and protein kinase activities and activates 3'-5'- exonuclease activity.

We have previously found that Drosophila melanogaster only has one deoxyribonucleoside kinase, Dm-dNK, however, capable to phosphorylate all four natural deoxyribonucleosides. Dm-dNK was originally isolated from an embryonic cell line. We wanted to study the expression of Dm-dNK during development from embryonic cells to adult flies and found declining Dm-dNK activity during development and no activity in adult flies.

Non-Viral Deoxyribonucleoside Kinases--Diversity and Practical Use.

Deoxyribonucleoside kinases (dNKs) phosphorylate deoxyribonucleosides to their corresponding monophosphate compounds. dNks also phosphorylate deoxyribonucleoside analogues that are used in the treatment of cancer or viral infections. The study of the mammalian dNKs has therefore always been of great medical interest.

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.

Thymidine kinase 1 regulatory fine-tuning through tetramer formation.

Thymidine kinase 1 (TK1) provides a crucial precursor, deoxythymidine monophosphate, for nucleic acid synthesis, and the activity of TK1 increases by up to 200-fold during the S-phase of cell division in humans. An important part of the regulatory checkpoints is the ATP and enzyme concentration-dependent transition of TK1 from a dimer with low catalytic efficiency to a tetramer with high catalytic efficiency. This regulatory fine-tuning serves as an additional control to provide a balanced pool of nucleic acid precursors in the cell.

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 (EC 2.7.

Deoxyribonucleoside kinases in two aquatic bacteria with high specificity for thymidine and deoxyadenosine.

Deoxyribonucleoside kinases (dNKs) are essential in the mammalian cell but their 'importance' in bacteria, especially aquatic ones, is less clear. We studied two aquatic bacteria, Gram-negative Flavobacterium psychrophilum JIP02/86 and Polaribacter sp. MED152, for their ability to salvage deoxyribonucleosides (dNs).

Comparative active-site mutation study of human and Caenorhabditis elegans thymidine kinase 1.

The first step for the intracellular retention of several anticancer or antiviral nucleoside analogues is the addition of a phosphate group catalysed by a deoxyribonucleoside kinase such as thymidine kinase 1 (TK1). Recently, human TK1 (HuTK1) has been crystallized and characterized using different ligands. To improve our understanding of TK1 substrate specificity, we performed a detailed, mutation-based comparative structure-function study of the active sites of two thymidine kinases: HuTK1 and Caenorhabditis elegans TK1 (CeTK1).

Reversible tetramerization of human TK1 to the high catalytic efficient form is induced by pyrophosphate, in addition to tripolyphosphates, or high enzyme concentration.

Thymidine kinase (TK1) is a key enzyme in the salvage pathway of deoxyribonucleotide metabolism, catalyzing the first step in the synthesis of dTTP by transfer of a gamma-phosphate group from a nucleoside triphosphate to the 5'-hydroxyl group of thymidine, forming dTMP. Human TK1 is cytosolic and its activity is absent in resting cells, appears in late G(1), increases in S phase coinciding with the increase in DNA synthesis, and disappears during mitosis. The fluctuation of TK1 through the cell cycle is important in providing a balanced supply of dTTP for DNA replication, and is partly due to regulation of TK1 expression at the transcriptional level.

A multisubstrate deoxyribonucleoside kinase from plants.

Deoxyribonucleoside kinases catalyze the rate limiting step during the salvage of deoxyribonucleosides and convert them into the corresponding monophosphate compounds. We have identified and characterized a unique multisubstrate deoxyribonucleoside kinase from plants. The phylogenetic relationship and biochemical properties suggest that this deoxyribonucleoside kinase represents a living fossil resembling the progenitor of the modern animal deoxycytidine, deoxyguanosine and thymidine 2 kinases.

Structural studies of nucleoside analog and feedback inhibitor binding to Drosophila melanogaster multisubstrate deoxyribonucleoside kinase.

The Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (dNK; EC 2.7.1.

Mitochondria as determinant of nucleotide pools and chromosomal stability.

Mitochondrial function plays an important role in multiple human diseases and mutations in the mitochondrial genome have been detected in nearly every type of cancer investigated to date. However, the mechanism underlying the interrelation is unknown. We used human cell lines depleted of mitochondrial DNA as models and analyzed the outcome of mitochondrial dysfunction on major cellular repair activities.

Nucleoside analogues are activated by bacterial deoxyribonucleoside kinases in a species-specific manner.

Objectives: To investigate the bactericidal activity of antiviral and anticancer nucleoside analogues against a variety of pathogenic bacteria and characterize the activating enzymes, deoxyribonucleoside kinases (dNKs).

Methods: Several FDA-approved nucleoside analogue drugs were screened for their potential bactericidal activity against several clinical bacterial isolates and type strains. We identified and subcloned the genes coding for putative deoxyribonucleoside kinases in Escherichia coli, Pasteurella multocida, Salmonella enterica, Yersinia enterocolitica, Bacillus cereus, Clostridium perfringens and Listeria monocytogenes.

Functional studies of active-site mutants from Drosophila melanogaster deoxyribonucleoside kinase. Investigations of the putative catalytic glutamate-arginine pair and of residues responsible for substrate specificity.

The catalytic reaction mechanism and binding of substrates was investigated for the multisubstrate Drosophila melanogaster deoxyribonucleoside kinase. Mutation of E52 to D, Q and H plus mutations of R105 to K and H were performed to investigate the proposed catalytic reaction mechanism, in which E52 acts as an initiating base and R105 is thought to stabilize the transition state of the reaction. Mutant enzymes (E52D, E52H and R105H) showed a markedly decreased k(cat), while the catalytic activity of E52Q and R105K was abolished.

Structural basis for the changed substrate specificity of Drosophila melanogaster deoxyribonucleoside kinase mutant N64D.

The Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) double mutant N45D/N64D was identified during a previous directed evolution study. This mutant enzyme had a decreased activity towards the natural substrates and decreased feedback inhibition with dTTP, whereas the activity with 3'-modified nucleoside analogs like 3'-azidothymidine (AZT) was nearly unchanged. Here, we identify the mutation N64D as being responsible for these changes.

Structures of thymidine kinase 1 of human and mycoplasmic origin.

Cytosolic thymidine kinase 1, TK1, is a well known cell-cycle-regulated enzyme of importance in nucleotide metabolism as well as an activator of antiviral and anticancer drugs such as 3'-azido-3'-deoxythymidine (AZT). We have now determined the structures of the TK1 family, the human and Ureaplasma urealyticum enzymes, in complex with the feedback inhibitor dTTP. The TK1s have a tetrameric structure in which each subunit contains an alpha/beta-domain that is similar to ATPase domains of members of the RecA structural family and a domain containing a structural zinc.

Effect of valine 106 on structure-function relation of cytosolic human thymidine kinase. Kinetic properties and oligomerization pattern of nine substitution mutants of V106.

Information on the regulation and structure-function relation of enzymes involved in DNA precursor synthesis is pivotal, as defects in several of these enzymes have been found to cause depletion or deletion of mitochondrial DNA resulting in severe diseases. Here, the effect of amino acid 106 on the enzymatic properties of the cell-cycle-regulated human cytosolic thymidine kinase 1 (TK1) is investigated. On the basis of the previously observed profound differences between recombinant TK1 with Val106 (V106WT) and Met106 (V106M) in catalytic activity and oligomerization pattern, we designed and characterized nine mutants of amino acid 106 differing in size, conformation and polarity.

Animal deoxyribonucleoside kinases: 'forward' and 'retrograde' evolution of their substrate specificity.

Deoxyribonucleoside kinases, which catalyse the phosphorylation of deoxyribonucleosides, are present in several copies in most multicellular organisms and therefore represent an excellent model to study gene duplication and specialisation of the duplicated copies through partitioning of substrate specificity. Recent studies suggest that in the animal lineage one of the progenitor kinases, the so-called dCK/dGK/TK2-like gene, was duplicated prior to separation of the insect and mammalian lineages. Thereafter, insects lost all but one kinase, dNK (EC 2.

Ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates and related analogues to replace ATP as phosphate donor for all human and Drosphila melanogaster deoxyribonucleoside kinases.

Six non-conventional adenosine-2'- and 3'-triphosphate analogues of ATP were tested as potential phosphate donors for all four human, and D. melanogaster, deoxyribonucleoside kinases. With dCK (only dAdo as acceptor), TK1, TK2 and dNK only 3'-deoxyadenosine-2'-triphosphate was an effective donor (5-60% that for ATP).

Striking ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates and related analogues to replace ATP as phosphate donor for all four human, and the Drosophila melanogaster, deoxyribonucleoside kinases.

In extension of an earlier report, six non-conventional analogues of ATP, three adenosine-2'-triphosphates (3'-deoxy, 3'-deoxy-3'-fluoro- and 3'-deoxy-3'-fluoroxylo-), and three adenosine-3'-triphosphates (2'-deoxy-, 2'-deoxy-2'-fluoro- and 2'-deoxy-2'-fluoroara-), were compared with ATP as potential phosphate donors for human deoxycytidine kinase (dCK), cytosolic thymidine kinase (TK1), mitochondrial TK2, deoxyguanosine kinase (dGK), and the deoxyribonucleoside kinase (dNK) from Drosophila melanogaster. With one group of enzymes, comprising TK1, TK2, dNK and dCK (with dAdo as acceptor), only 3'-deoxyadenosine-2'-triphosphate was an effective donor (5-60% that for ATP), and the other five analogues much less so, or inactive. With a second set, including dCK (dCyd, but not dAdo, as acceptor) and dGK (dGuo as acceptor), known to share high sequence similarity (approximately 45% sequence identity), all six analogues were good to excellent donors (13-119% that for ATP).