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

Abstract

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...

Molecular and Functional Interactions between Escherichia coli Nucleoside-diphosphate Kinase and the Uracil-DNA Glycosylase Ung

“…It is notable that, although Ndk already has several established interacting partners in DNA metabolism (23,25), this is the first indication of Ndk acting as a regulator in a specific DNA repair pathway. More important, to our knowledge, this constitutes the first report of an interaction involving the E. coli Ung enzyme with another E. coli protein.…”

“…Section 1734 solely to indicate this fact. 1 and while providing nucleoside triphosphates for DNA and RNA synthesis, Ndk also plays a role in maintaining the pools of cellular nucleoside triphosphates (24,25). Mutants of E. coli lacking Ndk exhibit normal growth rates but display a mutator phenotype that cannot be entirely attributed to the lack of phosphotransferase activity (26) or to an imbalance in the cellular concentrations of nucleoside triphosphates (24,27).…”

Adenylate Kinase of Escherichia coli, a Component of the Phage T4 dNTP Synthetase Complex

“…Purification of Recombinant E. coli Adenylate Kinase-The pellet was resuspended in 40 mM Tris-HCl (pH 8.0), 20 mM ␤-mercaptoethanol containing leupeptin, pepstatin A, benzamidine, and aprotinin (11). Following sonication, the lysate was centrifuged at 92,000 ϫ g for 60 min.…”

“…Glutathione S-Transferase Pulldown Assays-GST fusion proteins were prepared, and GST pulldown experiments carried out, as described in previous publications from this laboratory (11,20). Fusion proteins made for this study included gp1 (deoxyribonucleoside monophosphate kinase), gp42 (dCMP hydroxymethylase), gpfrd (dihydrofolate reductase), and E. coli nucleoside diphosphate kinase.…”

“…E. coli NDP kinase interacts directly or indirectly with several T4 enzymes of dNTP synthesis and DNA replication, and we consider it an integral component of the dNTP synthetase complex (11). In an early study (12) our laboratory found about 5% of the total adenylate kinase activity in the cell to cosediment with enzymes of the dNTP synthetase complex; NDP kinase behaved similarly.…”