Tomato thymidine kinase is subject to inefficient TTP feedback regulation.

A promising suicide gene therapy system to treat gliomas has been reported: the thymidine kinase 1 from tomato (toTK1) combined with the nucleoside analog pro-drug zidovudine (azidothymidine, AZT), which is known to penetrate the blood-brain barrier. Transduction with toTK1 has been found to efficiently increase the sensitivity of human glioblastoma cells to AZT, and nude rats with intracranial glioblastoma grafts have shown significantly improved survival when treated with the toTK1/AZT system. We show in our paper that the strong suicidal effect of AZT together with toTK1 may be explained by reduced TTP-mediated feedback inhibition of the AZT phosphorylation.

A mathematical model of human thymidine kinase 2 activity.

The mitochondrial enzyme thymidine kinase 2 (TK2) phosphorylates deoxythymidine (dT) and deoxycytidine (dC) to form dTMP and dCMP, which in cells rapidly become the negative-feedback end-products dTTP and dCTP. TK2 kinetic activity exhibits Hill coefficients of ∼0.5 (apparent negative cooperativity) for dT and ∼1 for dC. We present a mathematical model of TK2 activity that is applicable if TK2 exists as two monomer forms in equilibrium.

Thymidine kinase 1 deficient cells show increased survival rate after UV-induced DNA damage.

Balanced deoxynucleotide pools are known to be important for correct DNA repair, and deficiency for some of the central enzymes in deoxynucleotide metabolism can cause imbalanced pools, which in turn can lead to mutagenesis and cell death. Here we show that cells deficient for the thymidine salvage enzyme thymidine kinase 1 (TK1) are more resistant to UV-induced DNA damage than TK1 positive cells although they have thymidine triphosphate (dTTP) levels of only half the size of control cells. Our results suggest that higher thymidine levels in the TK- cells caused by defect thymidine salvage to dTTP protects against UV irradiation.

Enzymatic regulation of cytosolic thymidine kinase 1 and mitochondrial thymidine kinase 2: a mini review.

The central enzyme on the de novo pathway for synthesis of DNA precursors, the deoxyribonucleoside triphosphates, is ribonucleotide reductase (RNR). Deoxythymidine triphosphate (dTTP) has a key role in control of RNR activity shifting the specificity from pyrimidine to purine nucleotide reduction. Apart from the complex de novo synthesis of dTTP through UDP reduction, dTTP is provided through salvage of thymidine catalyzed by the thymidine kinases, the cytosolic and cell cycle regulated TK1 and the mitochondrial and constitutively expressed TK2. The complex enzymatic regulation of TK1 and TK2 and the possible physiological significance of this regulation will be discussed.

Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs.

Transduced deoxyribonucleoside kinases (dNK) can be used to kill recipient cells in combination with nucleoside prodrugs. The Drosophila melanogaster multisubstrate dNK (Dm-dNK) displays a superior turnover rate and has a great plasticity regarding its substrates. We used directed evolution to create Dm-dNK mutants with increased specificity for several nucleoside analogs (NAs) used as anticancer or antiviral drugs. Four mutants were characterized for the ability to sensitize Escherichia coli toward analogs and for their substrate specificity and kinetic parameters. The mutants had a reduced ability to phosphorylate pyrimidines, while the ability to phosphorylate purine analogs was relatively similar to the wild-type enzyme. We selected two mutants, for expression in the osteosarcoma 143B, the glioblastoma U-87M-G and the breast cancer MCF7 cell lines. The sensitivities of the transduced cell lines in the presence of the NAs fludarabine (F-AraA), cladribine (CdA), vidarabine and cytarabine were compared to the parental cell lines. The sensitivity of 143B cells was increased by 470-fold in the presence of CdA and of U-87M-G cells by 435-fold in the presence of F-AraA. We also show that a choice of the selection and screening system plays a crucial role when optimizing suicide genes by directed evolution.

Thymidine kinase diversity in bacteria.

Thymidine kinases (TKs) appear to be almost ubiquitous and are found in nearly all prokaryotes, eukaryotes, and several viruses. They are the key enzymes in thymidine salvage and activation of several anti-cancer and antiviral drugs. We show that bacterial TKs can be subdivided into 2 groups. The TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria.

Purification and characterization of wild-type and mutant TK1 type kinases from Caenorhabditis elegans.

Caenorhabditis elegans has a single deoxynucleoside kinase-like gene. The sequence is similar to that of human TK1, but besides accepting thymidine as a substrate, the C. elegans TK1 (CeTK1) also phosphorylates deoxyguanosine. In contrast to human TK1, the CeTK1 exclusively exists as a dimer with a molecular mass of approximately 60 kDa, even if incubated with ATP. Incubation with ATP induces a transition into a more active enzyme with a higher kcat but unchanged Km. This activation only occurs at an enzyme concentration in the incubation buffer of 0.5 micro g/ml (8.42 nM) or higher. C-terminal deletion of the enzyme results in lower catalytic efficiency and stability.

The role of mitochondrial dNTP levels in cells with reduced TK2 activity.

Both the nuclear and mitochondrial DNA (mtDNA) depend on separate balanced pools of dNTPs for correct function of DNA replication and repair of DNA damage. Import of dNTPs from the cytosolic compartment to the mitochondria has been suggested to have the potential of rectifying a mitochondrial dNTP imbalance. Reduced TK2 activity has been demonstrated to result in mitochondrial dNTP imbalance and consequently mutations of mtDNA in non-dividing cells. In this study, the consequences of a reduced thymidine kinase 2 (TK2) activity were measured in proliferating HeLa cells, on both whole-cell as well as mitochondrial dNTP levels. With the exception of increased mitochondrial dCTP level no significant difference was found in cells with reduced TK2 activity. Our results suggest that import of cytosolic dNTPs in mitochondria of proliferating cells can compensate a TK2 induced imbalance of the mitochondrial dNTP pool.

Effect of C-terminal of human cytosolic thymidine kinase (TK1) on in vitro stability and enzymatic properties.

Thymidine kinase (TK1) is a key enzyme in the salvage pathway of nucleotide metabolism and catalyzes the first rate-limiting step in the synthesis of dTTP, transfer of a gamma-phosphate group from a nucleoside triphosphate to the 5'-hydroxyl group of thymidine, thus forming dTMP. TK1 is cytosolic and its activity fluctuates during cell cycle coinciding with the DNA synthesis rate and disappears during mitosis. This fluctuation is important for providing a balanced supply of dTTP for DNA replication.The cell cycle specific activity of TK1 is regulated at the transcriptional level, but posttranslational mechanisms seem to play an important role for the level of functional TK1 protein as well. Thus, the C-terminal of TK1 is known to be essential for the specific degradation of the enzyme at the G2/M phase. In this work, we have studied the effect of deletion of the C-terminal 20, 40, and 44 amino acids of TK1 on in vitro stability, oligomerization, and enzyme kinetics. We found that deletion of the C-terminal fold markedly increased the stability as well as the catalytic activity.

Structure and function of cellular deoxyribonucleoside kinases.

Deoxyribonucleoside kinases phosphorylate deoxyribonucleosides, a crucial reaction in biosynthesis of DNA precursors through the salvage pathway. Their medical interest stems from their activation of a number of anticancer and antiviral drugs such as 2-chloro-2'-deoxyadenosine, azidothymidine and acyclovir. Here we review what is presently known about each of the mammalian kinases as well as some other members of the deoxyribonucleoside kinase family. A description of the biochemical properties of the enzymes is followed by an overview of the structural studies made on this family of enzymes, including the catalytic mechanism as well as the mechanism for feedback inhibition. A presentation of homology models of other proteins in the family is made and, finally, the determinants of substrate and substrate analog specificities are described.

Structural basis for substrate specificities of cellular deoxyribonucleoside kinases.

Deoxyribonucleoside kinases phosphorylate deoxyribonucleosides and activate a number of medically important nucleoside analogs. Here we report the structure of the Drosophila deoxyribonucleoside kinase with deoxycytidine bound at the nucleoside binding site and that of the human deoxyguanosine kinase with ATP at the nucleoside substrate binding site. Compared to the human kinase, the Drosophila kinase has a wider substrate cleft, which may be responsible for the broad substrate specificity of this enzyme. The human deoxyguanosine kinase is highly specific for purine substrates; this is apparently due to the presence of Arg 118, which provides favorable hydrogen bonding interactions with the substrate. The two new structures provide an explanation for the substrate specificity of cellular deoxyribonucleoside kinases.

Collateral sensitivity to gemcitabine (2',2'-difluorodeoxycytidine) and cytosine arabinoside of daunorubicin- and VM-26-resistant variants of human small cell lung cancer cell lines.

Multidrug resistance (MDR), characterized by a cross-resistance to many natural toxin-related compounds, may be caused either by overexpression of a drug efflux pump such as P-glycoprotein, (P-gP), multidrug resistance proteins MRP1-3, or BCRP/MXR or, in the case of DNA topoisomerase II active drugs, by a decrease in the enzymatic activity of the target molecule termed altered topoisomerase MDR (at-MDR). However, human small cell lung carcinoma (SCLC) cell lines showed a collateral sensitivity to 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) and 1-beta-D-arabinofuranosylcytosine (ara-C). H69/DAU, a daunorubicin (DAU)-resistant variant of H69 with a P-gP overexpression, and NYH/VM, a VM-26 (teniposide)-resistant variant of NYH with an at-MDR, were both 2-fold more sensitive to gemcitabine and 7- and 2-fold more sensitive to ara-C, respectively. MDR variants had a 4.3- and 2.0-fold increased activity of deoxycytidine kinase (dCK), respectively. dCK catalyzes the first rate-limiting activation step of both gemcitabine and ara-C. In addition, deoxycytidine deaminase, responsible for inactivation of dFdC and ara-C, was 9.0-fold lower in H69/DAU cells. The level of thymidine kinase 2, a mitochondrial enzyme that can also phosphorylate deoxycytidine and gemcitabine, was not significantly different between the variants. These differences most likely caused an increased accumulation of the active metabolites (dFdCTP, 2.1- and 1.6-fold in NYH/VM and H69/DAU cells, respectively) and of ara-CTP (1.3-fold in NYH/VM cells). Ara-CTP accumulation was not detectable in either H69 variant. The pools of all ribonucleoside and deoxyribonucleoside triphosphates were at least 3- to 4-fold higher in the NYH variants compared to the H69 variants; for dCTP and dGTP this difference was even larger. The higher ribonucleotide pools might explain the >10-fold higher accumulation of dFdCTP in NYH compared to H69 variants. Since dCTP is low, H69 cells might not need a high ara-CTP accumulation to inhibit DNA polymerase. This might be related to the lack of ara-CTP in H69 variants. In addition, the increased CTP, ATP, and UTP pools in the MDR variants might explain the increased ara-CTP and dFdCTP accumulation. In conclusion, the MDR variants of the human SCLC cell lines were collaterally sensitive due to an increased dCK activity, and consequently an increased ara-CTP and dFdCTP accumulation.

Identification of residues involved in the specificity and regulation of the highly efficient multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster.

In contrast to all known deoxyribonucleoside kinases, a single highly efficient deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) is able to phosphorylate all precursor nucleosides for DNA synthesis. Dm-dNK was mutated in vitro by high-frequency random mutagenesis, expressed in the thymidine kinase-deficient Escherichia coli strain KY895 and clones were selected for sensitivity to the nucleoside analogs 1-beta-d-arabinofuranosylcytosine (AraC, Cytarabine), 3'-azido-2', 3'-dideoxythymidine (AZT, Zidovudine, Retrovir, 2', 3'-dideoxyadenosine (ddA) and 2',3'-dideoxycytidine (ddC, Zalcitabine, Hivid. Thirteen mutants with increased sensitivity compared to the wild-type Dm-dNK were isolated from a relatively small pool of less than 10,000 clones. Eight mutant Dm-dNKs increased the sensitivity of KY895 to more than one analog, and two of these mutants even to all four nucleoside analogs. Surprisingly, the mutations did not map to the five regions which are highly conserved among deoxyribonucleoside kinases. The molecular background of improved sensitivity was characterized for the double-mutant MuD (N45D, N64D), where the LD(100) value of transformed KY895 decreased 316-fold for AZT and more than 11-fold for ddC when compared to wild-type Dm-dNK. Purified recombinant MuD displayed higher K(m) values for the native substrates than wild-type Dm-dNK and the V(max) values were substantially lower. On the other hand, the K(m) and V(max) values for AZT and the K(m) value for ddC were nearly unchanged between MuD and wild-type Dm-dNK. Additionally, a decrease in feedback inhibition of MuD by thymidine triphosphate (TTP) was found. This study demonstrates how high-frequency mutagenesis combined with a parallel selection for desired properties provides an insight into the structure-function relationships of the multisubstrate kinase from D. melanogaster. At the same time these mutant enzymes exhibit properties useful in biotechnological and medical applications.

Valine, not methionine, is amino acid 106 in human cytosolic thymidine kinase (TK1). Impact on oligomerization, stability, and kinetic properties.

Cytosolic thymidine kinase (TK1) cDNA from human lymphocytes was cloned, expressed in Escherichia coli, purified, and characterized with respect to the ATP effect on thymidine affinity and oligomerization. Sequence analysis of this lymphocyte TK1 cDNA and 21 other cDNAs or genomic TK1 DNAs from healthy cells or leukemic or transformed cell lines revealed a valine at amino acid position 106. The TK1 sequence in NCBI GenBank(TM) has methionine at this position. The recombinant lymphocyte TK1(Val-106) (rLy-TK1(Val-106)) has the same enzymatic and oligomerization properties as endogenous human lymphocyte TK1 (Ly-TK1); ATP exposure induces an enzyme concentration-dependent reversible transition from a dimer to a tetramer with 20-30-fold higher thymidine affinity (K(m) about 15 and 0.5 microm, respectively). Substitution of Val-106 with methionine to give rLy-TK1(Met-106) results in a permanent tetramer with the high thymidine affinity (K(m) about 0.5 microm), even without ATP exposure. Furthermore, rLy-TK1(Met-106) is considerably less stable than rLy-TK1(Val-106) (t(12) at 15 degrees C is 41 and 392 min, respectively). Because valine with high probability is the naturally occurring amino acid at position 106 in human TK1 and because this position has high impact on the enzyme properties, the Val-106 form should be used in future investigations of recombinant human TK1.

Functional expression of a multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster and its C-terminal deletion mutants.

The occurrence of a deoxyribonucleoside kinase in Drosophila melanogaster (Dm-dNK) with remarkably broad substrate specificity has recently been indicated (Munch-Petersen, B., Piskur, J., and Søndergaard, L. (1998) J. Biol. Chem. 273, 3926-3931). To prove that the capacity to phosphorylate all four deoxyribonucleosides is in fact associated to one polypeptide chain, partially sequenced cDNA clones, originating from the Berkeley Drosophila genome sequencing project, were searched for homology with human deoxyribonucleoside kinases. The total sequence of one cDNA clone and the corresponding genomic DNA was determined and expressed in Escherichia coli as a glutathione S-transferase fusion protein. The purified and thrombin cleaved recombinant protein phosphorylated the four deoxyribonucleosides with high turnover and K(m) values similar to those of the native Dm-dNK, as well as the four ribonucleosides and many therapeutical nucleoside analogs. Dm-dNK has apparently the same origin as the mammalian kinases, thymidine kinase 2, deoxycytidine kinase, deoxyguanosine kinase, and the herpes viral thymidine kinases, but it has a unique C terminus that seems to be important for catalytic activity and specificity. The C-terminal 20 amino acids were dispensable for phosphorylation of deoxyribonucleosides but necessary for full activity with purine ribonucleosides. Removal of the C-terminal 20 amino acids increased the specific activity 2-fold, but 99% of the activity was lost after removal of the C-terminal 30 amino acids.

Substrate/inhibitor properties of human deoxycytidine kinase (dCK) and thymidine kinases (TK1 and TK2) towards the sugar moiety of nucleosides, including O'-alkyl analogues.

Nucleoside analogues with modified sugar moieties have been examined for their substrate/inhibitor specificities towards highly purified deoxycytidine kinase (dCK) and thymidine kinases (tetrameric high-affinity form of TK1, and TK2) from human leukemic spleen. In particular, the analogues included the mono- and di-O'-methyl derivatives of dC, dU and dA, syntheses of which are described. In general, purine nucleosides with modified sugar rings were feebler substrates than the corresponding cytosine analogues. Sugar-modified analogues of dU were also relatively poor substrates of TK1 and TK2, but were reasonably good inhibitors, with generally lower Ki values vs TK2 than TK1. An excellent discriminator between TK1 and TK2 was 3'-hexanoylamino-2',3'-dideoxythymidine, with a Ki of approximately 600 microM for TK1 and approximately 0.1 microM for TK2. 3'-OMe-dC was a superior inhibitor of dCK to its 5'-O-methyl congener, consistent with possible participation of the oxygen of the (3')-OH or (3')-OMe as proton acceptor in hydrogen bonding with the enzyme. Surprisingly alpha-dT was a good substrate of both TK1 and TK2, with Ki values of 120 and 30 microM for TK1 and TK2, respectively; and a 3'-branched alpha-L-deoxycytidine analogue proved to be as good a substrate as its alpha-D-counterpart. Several 5'-substituted analogues of dC were good non-substrate inhibitors of dCK and, to a lesser extent, of TK2. Finally, some ribonucleosides are substrates of the foregoing enzymes; in particular C is a good substrate of dCK, and 2'-OMe-C is an even better substrate than dC.

Human thymidine kinase 2: molecular cloning and characterisation of the enzyme activity with antiviral and cytostatic nucleoside substrates.

Based on amino acid sequence information from purified mitochondrial thymidine kinase (TK2), a cDNA of 1930 bp was cloned, containing an open reading frame encoding 232 amino acid residues starting with the N-terminal sequence determined from the native human protein preparation. Northern blot analysis with the cDNA coding region demonstrated several TK2 mRNAs, with 2 and 4 kb forms present in many tissues. We also characterised N-terminally truncated (starting at position 18) human TK2 with pharmacologically important antiviral and cytostatic nucleoside analogues. Results were highly similar to those with the native TK2 preparation. The anti-leukaemic drug arabinosyl cytosine is phosphorylated. The antitumour drug difluorodeoxycytidine and its metabolite difluorodeoxyuridine are good substrates, with K(m) values of 66 and 29 microM, respectively, and a relative Vmax of 0.6 compared to that of thymidine. Negative cooperativity was found with thymidine and the anti-HIV drug 3'-azidothymidine, but the reaction followed Michaelis-Menten kinetics with deoxycytidine, arabinosyl cytosine, and arabinosyl thymine. The results demonstrate a broad substrate specificity and complex kinetics, and suggest a role for TK2 in the activation of chemotherapeutic nucleoside analogues.