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

Abstract

Adenylate kinase, which catalyzes the reversible ATPdependent phosphorylation of AMP to ADP and dAMP to dADP, can also catalyze the conversion of nucleoside diphosphates to the corresponding triphosphates. Lu and Inouye (Lu, Q., and Inouye, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5720 -5725) showed that an Escherichia coli ndk mutant, lacking nucleoside diphosphate kinase, can use adenylate kinase as an alternative source of nucleoside triphosphates. Bacteriophage T4 can reproduce in an Escherichia coli ndk mutant, implying that adenylate kinase can meet a demand for deoxyribonucleoside triphosphates that increases by up to 10-fold as a result of T4 infection. In terms of kinetic linkage and specific protein-protein associations, NDP kinase is an integral component of T4 dNTP synthetase, a multienzyme complex containing phage-coded enzymes, which facilitates the synthesis of dNTPs and their flow into DNA. Here we asked whether, by similar criteria, adenylate kinase of the host cell is also a specific component of the complex. Experiments involving protein affinity chromatography, immunoprecipitation, optical biosensor measurements, and glutathione S-transferase pulldowns demonstrated direct interactions between adenylate kinase and several phagecoded enzymes, as well as E. coli nucleoside diphosphate kinase. These results identify adenylate kinase as a specific component of the complex. The rate of DNA synthesis after infection of an ndk mutant was found to be about 40% of the rate seen in wild-type infection, implying that complementation of the missing NDP kinase function by adenylate kinase is fairly efficient, but that adenylate kinase becomes rate-limiting for DNA synthesis when it is the sole source of dNTPs.Adenylate kinase catalyzes the reversible ATP-dependent phosphorylation of AMP to ADP. The reaction is involved in the de novo biosynthesis of adenine nucleotides, and it is also thought to participate in adjusting adenine nucleotide levels to meet the energy demands of a cell (1). In addition, there is evidence that the enzyme in Escherichia coli participates in phospholipid biosynthesis, although the specific involvement has not been defined (2). Temperature-sensitive mutations in adk, the structural gene for adenylate kinase, cause defective phospholipid synthesis when bacteria are grown at a nonpermissive temperature (3).A novel capability for E. coli adenylate kinase was described when Lu and Inouye (4) found that the wild-type adk gene could complement a site-specific disruption of ndk, the structural gene for nucleoside diphosphate kinase. Lu and Inouye (5) showed that adenylate kinase could catalyze the conversion of nucleoside diphosphates to triphosphates. Experiments in our laboratory (5) showed that the phosphate donor for at least some of these reactions was ADP. So, instead of catalyzing the well known reaction, 2ADP º AMP ϩ ATP, the enzyme was evidently substituting a different nucleoside diphosphate for one of the two ADPs:Both NDP 1 kinase and adenylate kinase were shown, some ye...

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.…”

“…coli Ndk is also multifunctional; it can directly interact with a half dozen other proteins involved in DNA metabolism (22,23) and has been found in a DNA replication complex (23),…”

“…coli Ndk is also multifunctional; it can directly interact with a half dozen other proteins involved in DNA metabolism (22,23) and has been found in a DNA replication complex (23), * This work was supported by Grant ESO6070 (to G. P. P.) from the National Institutes of Health and by Grant RO1CA076496 (to E. H. P.) from NCI, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges.…”

“…E. coli NDP kinase is encoded by a single gene, ndk (4), whereas the genetically distinct forms of human NDP kinases are encoded by multiple genes termed NM23-H1 through H8 (5). The name NM (nonmetastatic) 23 was initially accorded to the matriarch of the family, NM23-H1, on the basis of its reported action as a tissue-specific metastasis inhibitor (6). Moreover, there is evidence that NM23/NDK proteins promote tumor formation and play various roles in normal development and cellular proliferation (7,8).…”

Defective Ribonucleoside Diphosphate Reductase Impairs Replication Fork Progression inEscherichia coli

“…Mathews and coworkers proposed, and have extensively described, the association of the nucleotide metabolism enzymes in a dNTP-synthesizing complex, explaining channeling of the biosynthesis and compartmentation of the precursors in T4 metabolism (13,(17)(18)(19)27). Recently, new approaches supporting the associations between dNTP synthesis enzymes, DNA, and the replication complex in T4-infected cells (14) and in E. coli (10,24) have been described.…”