(t1 ⁄2 at 15°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.The human cytosolic thymidine kinase, TK1 (EC 2.7.1.21), is a key enzyme in the salvage synthesis of TMP from thymidine. Intracellular TMP is quickly phosphorylated to TDP and TTP. Because TTP is an allosteric effector of ribonucleotide reductase, imbalances in the TTP pool disturb the supply of both purines and pyrimidines for DNA synthesis and repair. In turn, imbalanced deoxynucleoside triphosphate (dNTP) pools increase the mutation rate and probability of carcinogenesis (1-3).TK1 is a cell cycle-regulated enzyme. Its activity fluctuates with DNA synthesis, being high in dividing and malignant cells and low in quiescent cells (4, 5). The expression of TK1 is meticulously controlled on the transcriptional and post-transcriptional level (6, 7). At the enzymatic level, ATP, besides being a cosubstrate, has been shown to be a regulator of the catalytic activity of TK1 (8). Thus, exposure to ATP induces a reversible enzyme concentration-dependent transition from a low thymidine affinity dimer of about 50 kDa (K m ϭ 15 M) to a high affinity tetramer (K m ϭ 0.7 M). To further investigate the effect of ATP, we constructed a pET3a-TK1 plasmid (9) containing the amino acid coding region of TK1 cDNA from the pTK11 plasmid of Bradshaw and Deininger (10), who had used SV40 transformed human fibroblasts as the mRNA source. We expressed the resulting recombinant TK1 (rTFi-TK1) in Escherichia coli, and purified and characterized the enzyme. To our surprise we found that the enzymatic properties of rTFi-TK1 differed markedly from those of the endogenous Ly-TK1 with respect to the regulatory effect of ATP (9). Irrespective of preexposure to ATP, the recombinant rTFi-TK1 was a permanent tetramer of about 100 kDa with high affinity to thymidine with a K m value about 0.4 M (9). At that time, we assumed that the amino acid sequences of rTFi-TK1 and Ly-TK1 were identical and explained the divergent properties of rTFi-TK1 by the absence of post-translational modification of TK1 when expressed in E. coli (9). Because the pET3a-TK1 expression system was not satisfactory in terms of amount of TK1 protein produced, we constructed another expression plasmid, pGEX-2T-LyTK1. Here, the amino acid coding region of TK1 from normal human lymphocytes was cloned into the pGEX-2T vector, and the recombinant TK1 was expressed as an isopropyl-1-thio-␤-D-galactopyranoside-inducible glutathione S-transferase fusion protein. In contrast to the findings with rTFi-TK1, our preliminary kinetic experiments showed that the recombinant lymphocyte TK1 (rLy-TK1) 1 behaved essentially as the endogenous lymphocyte enzyme, Ly-TK1, regarding kinetic and oligomerization properties. Therefore, absence of posttranslational modification of rTFi-TK1 in E. coli cannot ex...

The Vibrionaceae family is distantly related to Enterobacteriaceae within the group of bacteria possessing the Dam methylase system. We have cloned, sequenced, and analyzed the dnaA gene region of Vibrio harveyi and found that although the organization of the V. harveyi dnaA region differs from that of Escherichia coli, the expression of both genes is autoregulated and ATP-DnaA binds cooperatively to ATP-DnaA boxes in the dnaA promoter region. The DnaA proteins of V. harveyi and E. coli are interchangeable and function nearly identically in controlling dnaA transcription and the initiation of chromosomal DNA replication despite the evolutionary distance between these bacteria.The DnaA initiator protein and the origin of replication, oriC, with its binding sites for the DnaA protein, the DnaA boxes, are the key elements in controlling the initiation of chromosomal DNA replication (15,23,29). The dnaA operon in Escherichia coli is transcribed from two promoters with a DnaA box located between them (16). dnaA transcription is autoregulated (1,5,19) Several chromosomal origins of replication from Enterobacteriaceae (9) and the origin from Vibrio harveyi-a luminous, motile, and facultatively anaerobic gram-negative rod belonging to the Vibrionaceae-are all functional in E. coli (42), whereas the Pseudomonas putida origin is not (40). In parallel, several dnaA genes from Enterobacteriaceae complement dnaA(Ts) mutations in E. coli (31, 32) but the P. putida dnaA gene does not (17). In the ␥ subdivision of Proteobacteria the families Enterobacteriaceae, Pasteurellaceae, and Vibrionaceae are distinguished from Pseudomonadaceae (10, 26) by having the Dam methylase system in common (4). The Dam methylase methylates the adenine in GATC sequences and plays a role in DNA replication, in mismatch repair, and in gene regulation (27). Overrepresentation of GATC sequences in the oriC and dnaA regions is a conserved feature in E. coli (22,34), in other Enterobacteriaceae (9, 31), and in V. harveyi (Vibrionaceae) (42). It takes a much longer time in E. coli to remethylate GATC sites within the clusters located in the oriC and the dnaA regions than other GATC sites. This delay of remethylation is due to sequestration of the GATC sites, most likely to the cell membrane (7,25). The period with the sequestered origins (the eclipse period) is important for the synchrony of initiation (15), and the SeqA protein (21, 37) is an absolute requisite for the eclipse period (38).We sought an evolutionarily distant dnaA gene that is functional in E. coli. Because neither the dnaA P. putida nor dnaA Haemophilus influenzae gene complements the dnaA46 mutation in E. coli (17; O. Skovgaard, unpublished data), we chose a member of Vibrionaceae, V. harveyi, for this purpose. We investigated whether the basic elements in the control of dnaA transcription regulation and DNA replication in V. harveyi are similar to those of E. coli. After cloning and physical and physiological analysis of the V. harveyi dnaA gene region, we conclude that the basic eleme...

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.

Deoxyribonucleoside kinases (dNKs; EC 2.7.1.145) catalyze the initial, and usually rate-determining step in the synthesis of the four DNA precursors (dNTPs) through the salvage pathway. These enzymes transfer the c-phosphoryl group from ATP to deoxyribonucleosides (dN) and form the corresponding dNMPs [1]. In the cell, dNMPs are quickly phosphorylated to dNDPs and dNTPs by ubiquitous mono-and diphosphate deoxyribonucleoside kinases.Deoxyribonucleoside kinases are also responsible for activation (initial phosphorylation) of nontoxic nucleoside analogs such as azidothymidine (AZT) and acyclovir (ACV) used in the treatment of cancer and viral diseases. After further phosphorylation by other cellular kinases the triphosphorylated nucleoside analogs are incorporated into DNA and cause chain termination and cell death [2]. Alternatively, they inhibit the DNA synthesizing machinery or initiate apoptosis [3]. 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. Furthermore, we crystallized the mutant enzyme in the presence of one of its substrates, thymidine, and the feedback inhibitor, dTTP. The introduction of the charged Asp residue appears to destabilize the LID region (residues 167-176) of the enzyme by electrostatic repulsion and no hydrogen bond to the 3¢-OH is made in the substrate complex by Glu172 of the LID region. This provides a binding space for more bulky 3¢-substituents like the azido group in AZT but influences negatively the interactions between Dm-dNK, substrates and feedback inhibitors based on deoxyribose. The detailed picture of the structure-function relationship provides an improved background for future development of novel mutant suicide genes for Dm-dNK-mediated gene therapy.

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. According to their oligomerization pattern and thymidine kinetics, the TK1 mutants can be divided into two groups. Group I (V106A, V106I and V106T) behaves like V106WT, in that pre-assay exposure to ATP induces reversible transition from a dimer with low catalytic activity to a tetramer with high catalytic activity. Group II (V106G, V106H, V106K, V106L and V106Q) behaves like V106M in that they are permanently high activity tetramers, irrespective of ATP exposure. We conclude that size and conformation of amino acid 106 are more important than polarity for the catalytic activity and oligomerization of TK1. The role of amino acid 106 and the sequence surrounding it for dimer-tetramer transition was confirmed by cloning the putative interface fragment of human TK1 and investigating its oligomerization pattern.Keywords: dimer-tetramer formation; enzyme kinetics; enzyme mutants; structure-function relation; thymidine kinase.Enzymes involved in salvage and metabolism of deoxynucleosides have an important role in the regulation of DNA precursors for DNA synthesis and repair. Recently, severe syndromes, such as mitochondrial neurogastrointestinal encephalomyopathy and mitochondrial DNA depletion syndrome which lead to multiple mitochondrial DNA abnormalities, were found to be caused by defects in the cytoplasmic thymidine phosphorylase [1,2] or the two mitochondrial deoxynucleoside kinases: deoxyguanosine kinase (dGK) and thymidine kinase 2 (TK2) respectively [3,4]. In contrast with earlier work suggesting spatial and metabolic separation of thymidine phosphate pools between the cytosol and mitochondria [5,6], recent evidence suggests the two compartments are connected by a rapid and dynamic exchange [7]. These findings may explain why defects in deoxynucleotide metabolic enzymes, mitochondrial as well as cytoplasmic, lead to severe mitochondrial DNA abnormality syndromes. Therefore, it is of great importance to acquire detailed knowledge about the properties of the enzymes involved in balancing the cellular and mitochondrial dNTP pools.Human cytosolic thymidine kinase (TK1; EC 2.7.1.21) is a salvage pathway enzyme in the synthesis of the DNA precursor dTTP. It catalyzes the first step of this pathway, in which thymidine is phosphorylated to dTMP [8]. In turn, intracellular dTMP is rapidly phosphorylated to dTTP, an allosteric effector of rib...