T4 Phage deoxyribonucleoside triphosphate synthetase: Purification of an enzyme complex and identification of gene products required for integrity

Moen, Laura K.; Howell, Meredith L.; Lasser, Gerald W.; Mathews, Christopher K.

doi:10.1002/jmr.300010109

Cited by 28 publications

(12 citation statements)

References 22 publications

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“…Therefore, it is perhaps not surprising that the enzyme behaves as if associated with the dNTP synthetase complex, as shown both by sedimentation analysis (4, 7) and kinetic experiments on catalysis of multi-step reaction pathways (4,8). Moreover, the enzyme associates, directly or indirectly, with about a half-dozen phage-coded proteins of dNTP and DNA metabolism, as shown by analysis of T4 proteins retained on columns of immobilized recombinant NDP kinase (9).…”

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confidence: 99%

“…Our laboratory (4) and that of G. R. Greenberg (5) have proposed that the enzymes of dNTP synthesis form a multienzyme complex and that this "dNTP synthetase" complex is juxtaposed with the replisome, so as to allow direct channeling of dNTPs to replication sites. Considerable evidence supports this model (7)(8)(9). 2 If the model is correct, then E. coli NDP kinase, which catalyzes the last reaction in dNTP synthesis, occupies a strategic position in the flow of nucleotides from the dNTP synthetase complex to the associated replisome.…”

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confidence: 99%

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Escherichia coli Nucleoside Diphosphate Kinase Interactions with T4 Phage Proteins of Deoxyribonucleotide Synthesis and Possible Regulatory Functions

Shen¹,

Olcott²,

Kim³

et al. 2004

Journal of Biological Chemistry

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In both prokaryotic and eukaryotic organisms, nucleoside diphosphate kinase is a multifunctional protein, with well defined functions in ribo-and deoxyribonucleoside triphosphate biosynthesis and more recently described functions in genetic and metabolic regulation, signal transduction, and DNA repair. This paper concerns two unusual properties of nucleoside diphosphate (NDP) kinase from Escherichia coli: 1) its ability to interact specifically with enzymes encoded by the virulent bacteriophage T4 and 2) its roles in regulating metabolism of the host cell. By means of optical biosensor analysis, fluorescence spectroscopy, immunoprecipitation, and glutathione S-transferase pull-down assays, we have shown that E. coli NDP kinase interacts directly with T4 thymidylate synthase, aerobic ribonucleotide reductase, dCTPase-dUTPase, gene 32 single-strand DNA-binding protein, and deoxycytidylate hydroxymethylase. The interactions with ribonucleotide reductase and with gp32 are enhanced by nucleoside triphosphates, suggesting that the integrity of the T4 dNTP synthetase complex in vivo is influenced by the composition of the nucleotide pool. The other investigations in this work stem from the unexpected finding that E. coli NDP kinase is dispensable for successful T4 phage infection, and they deal with two observations suggesting that the NDP kinase protein plays a genetic role in regulating metabolism of the host cell: 1) the elevation of CTP synthetase activity in an ndk mutant, in which the structural gene for NDP kinase is disrupted, and 2) the apparent ability of NDP kinase to suppress anaerobic growth in a pyruvate kinase-negative E. coli mutant. Our data indicate that the regulatory roles are metabolic, not genetic, in nature.Like other large virulent DNA phages, bacteriophage T4 encodes nearly all of the enzymes and proteins needed not only for its own DNA replication but for biosynthesis of the four deoxyribonucleoside triphosphates. The enzymes of dNTP 1 synthesis play two functions in T4 infection: 1) replacing the dCTP pool by 5-hydroxymethyl-dCTP, which gives rise to the novel phage-specific pyrimidine DNA base, 5-hydroxymethylcytosine, and 2) increasing the total rate of dNTP synthesis, necessary to sustain DNA accumulation at rates up to 10-fold higher per cell than the preinfection rate.In 1963 Bello and Bessman (1) showed that the level of one dNTP-synthesizing enzyme, nucleoside diphosphate kinase, did not change appreciably after T4 infection. As studied in extracts of T4-infected Escherichia coli, the specific activity of NDP kinase in either uninfected or infected E. coli is much higher than that of the phage-coded enzyme deoxyribonucleoside 5Ј-monophosphate kinase (gp1), which converts dNMPs to the dNDP substrates used by NDP kinase for dNTP synthesis (2, 3). This suggests that NDP kinase of the host cell carries out the conversion of dNDPs to dNTPs for phage DNA synthesis. That the activity of NDP kinase is so high should perhaps not be surprising, because the enzyme is also responsible for ribonucl...

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confidence: 99%

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confidence: 99%

Escherichia coli Nucleoside Diphosphate Kinase Interactions with T4 Phage Proteins of Deoxyribonucleotide Synthesis and Possible Regulatory Functions

Shen¹,

Olcott²,

Kim³

et al. 2004

Journal of Biological Chemistry

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“…The biological significance of binding to the latter three proteins on the list is unclear, although we note that earlier experiments from our laboratory (Moen et al, 1988) suggested an essential role for the regA gene product in assembly of the dNTP synthetase complex. Note that, although we might have expected to see dCMP deaminase among the bound proteins, we did not see a spot on the electrophoretogram that could be identified as the deaminase.…”

Section: Analysis Of Immobilized T4 Thymidylate Synthase-mentioning

confidence: 78%

“…3, because all of the reactions catalyzed are closely related, as shown below. A second reason for interest in these results is the fact that T4 dCMP deaminase and dTMP synthase are kinetically linked (Moen et al, 1988). When a preparation of the dNTP synthetase complex is provided with dCTP as a substrate, dTMP is produced with no detectable lag, indicating kinetic facilitation of the following reaction sequence, dCTP3dCMP3dUMP3dTMP.…”

Section: Discussionmentioning

confidence: 99%

Protein-Protein Interactions Involving T4 Phage-coded Deoxycytidylate Deaminase and Thymidylate Synthase

McGaughey

Wheeler

Moore

et al. 1996

Journal of Biological Chemistry

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The enzymes deoxycytidylate deaminase (EC 3.5.4.12) and thymidylate synthase (EC 2.1.1.45) are functionally associated with one another, since they catalyze sequential reactions. In T4 coliphage infection the two enzymes are found in dNTP synthetase, a multienzyme complex for deoxyribonucleotide biosynthesis. Protein-protein interactions involving the phage-coded forms of these two enzymes have been explored in three experiments that use the respective purified protein as an affinity ligand. First, an extract of radiolabeled T4 proteins was passed through a column of immobilized enzyme (either dTMP synthase or dCMP deaminase), and the specifically bound proteins were identified. Second, two mutant form of dCMP deaminase (H90N and H94N), altered in presumed zinc-binding sites, were analyzed similarly, with the results suggesting that some, but not all, interactions require normal structure near the catalytic site. Third, affinity chromatography using either enzyme as the immobilized ligand, revealed interactions between the two purified enzymes in the absence of other proteins. In these experiments we noted a significant effect of dCTP, an allosteric modifier of dCMP deaminase, upon the interactions.

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“…Elsewhere, however, we have described conditions that disrupt the dNTP synthetase complex (15), although with resultant decreases in DNA synthetic rates. Probably, the dNTP synthetase complex, while helping to maximize viral DNA synthesis, is dispensable, as long as each of the essential individual enzyme activities is present.…”

Section: Discussionmentioning

confidence: 99%

Effects of T4 phage infection and anaerobiosis upon nucleotide pools and mutagenesis in nucleoside diphosphokinase-defective Escherichia coli strains

et al. 1996

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Bacteriophage T4 encodes nearly all of its own enzymes for synthesizing DNA and its precursors. An exception is nucleoside diphosphokinase (ndk gene product), which catalyzes the synthesis of ribonucleoside triphosphates and deoxyribonucleoside triphosphates (dNTPs) from the corresponding diphosphates. Surprisingly, an Escherichia coli ndk deletion strain grows normally and supports T4 infection. As shown elsewhere, these ndk mutant cells display both a mutator phenotype and deoxyribonucleotide pool abnormalities. However, after T4 infection, both dNTP pools and spontaneous mutation frequencies are near normal. An E. coli strain carrying deletions in ndk and pyrA and pyrF, the structural genes for both pyruvate kinases, also grows and supports T4 infection. We examined anaerobic E. coli cultures because of reports that in anaerobiosis, pyruvate kinase represents the major route for nucleoside triphosphate synthesis in the absence of nucleoside diphosphokinase. The dNTP pool imbalances and the mutator phenotype are less pronounced in the anaerobic than in the corresponding aerobic ndk mutant strains. Anaerobic dNTP pool data, which have not been reported before, reveal a disproportionate reduction in dGTP, relative to the other pools, when aerobic and anaerobic conditions are compared. The finding that mutagenesis and pool imbalances are mitigated in both anaerobic and T4-infected cultures provides strong, if circumstantial, evidence that the mutator phenotype of ndk mutant cells is a result of the dNTP imbalance. Also, the viability of these cells indicates the existence of a second enzyme system in addition to nucleoside diphosphokinase for nucleoside triphosphate synthesis.Nucleoside diphosphokinase catalyzes transfer of the ␥-phosphate of nucleoside triphosphates to a wide variety of ribonucleoside diphosphates and deoxyribonucleoside diphosphates. Because the enzyme is equilibrium controlled and of low specificity, its action allows ATP, the most abundant nucleoside triphosphate in most cells, to serve as phosphate donor for the synthesis of the seven other common ribonucleoside triphosphates (rNTPs) and deoxyribonucleoside triphosphates (dNTPs). In T-even bacteriophage infection, NDP kinase catalyzes the only known reaction essential to DNA precursor biosynthesis that does not involve a virus-coded enzyme (see reference 14). NDP kinase of the host, Escherichia coli, is sufficiently active that it can support dNTP synthesis, even though demand increases by up to 10-fold after T4 infection, with a commensurate increase in the rate of DNA synthesis.NDP kinase is of particular interest in connection with T4 phage infection, because it interacts specifically with a number of T4-coded proteins and forms part of a multienzyme complex, called dNTP synthetase, which facilitates the synthesis of DNA precursors and, possibly, their transfer to the replication machinery (13,19). For this reason, we were surprised to learn that T4 can replicate in an E. coli strain bearing an engineered deletion mutation affecting ndk, ...

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T4 Phage deoxyribonucleoside triphosphate synthetase: Purification of an enzyme complex and identification of gene products required for integrity

Cited by 28 publications

References 22 publications

Escherichia coli Nucleoside Diphosphate Kinase Interactions with T4 Phage Proteins of Deoxyribonucleotide Synthesis and Possible Regulatory Functions

Escherichia coli Nucleoside Diphosphate Kinase Interactions with T4 Phage Proteins of Deoxyribonucleotide Synthesis and Possible Regulatory Functions

Protein-Protein Interactions Involving T4 Phage-coded Deoxycytidylate Deaminase and Thymidylate Synthase

Effects of T4 phage infection and anaerobiosis upon nucleotide pools and mutagenesis in nucleoside diphosphokinase-defective Escherichia coli strains

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