The current model of immune activation in bacteria. Various fungi stimulated antimicrobial peptides through at least two different pathways requiringRelish and/or Dif. Induction of Attacin A by Geotrichum candidum required Relish, whereas activation by Beauvaria bassiana required Dif, suggesting that the Drosophila immune system can distinguish between at least these two fungi. We conclude that the Drosophila immune system is more complex than the current model. We propose a new model to account for this immune system complexity, incorporating distinct pattern recognition receptors of the Drosophila immune system, which can distinguish between various fungi and G ؉ bacteria, thereby leading to selective induction of antimicrobial peptides via differential activation of Relish and Dif.

Two different analogs of ATP, [gamma-32P]2N3ATP and and [gamma-32P]8N3ATP, were used to photoaffinity label the MM and BB isoforms of rabbit cytosolic creatine kinase. Evidence that photoinsertion was within the ATP-binding domain was as follows: (1) Assays for creatine phosphate production demonstrated that [gamma-32]2N3ATP and [gamma-32P]8N3ATP are substrates for creatine kinase. (2) Enzymatic activity was inhibited by photolabeling with either analog. (3) Saturation of photoinsertion was observed for both analogs. Half-maximal saturation was observed at 5 microM [gamma-32P]2N3ATP or 12 microM (gamma-32P]8N3ATP. (4) Photoinsertion of both probes could be decreased by micromolar levels of ATP. Immobilized Al3+ affinity chromatography and HPLC were used to isolate the peptides modified by these probes. Overlapping sequence analysis of the isolated peptides from the tryptic and chymotryptic digests of the photolabeled MM isoform revealed that [gamma-32P]8N3ATP photoinserted into the peptide region corresponding to Val279-Arg291, whereas [gamma-32P]2N3-ATP photoinserted into Val236-Lys241. The corresponding peptide (Ile279-Arg291 and Val236-Lys241) from the BB isoform were shown to be selectively modified. We conclude that amino acid residues within the peptide regions 236-241 and 279-291 of rabbit cytosolic creatine kinase are localized within the binding domain for the adenine moiety of ATP. The results also demonstrate the effectiveness and selectivity of Al3+ as the chelating agent in immobilized metal affinity chromatography for the isolation of photolabeled peptides as well as its potential to enhance retention of radiolabel during HPLC.

No 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...

This article summarizes research from our laboratory on two aspects of the biochemistry of nucleoside diphosphate kinase from Escherichia coli--first, its interactions with several T4 bacteriophage-coded enzymes, as part of a multienzyme complex for deoxyribonucleoside triphosphate biosynthesis. We identify some of the specific interactions and discuss whether the complex is linked physically or functionally with the T4 DNA replication machinery, or replisome. Second, we discuss phenotypes of an E. coli mutant strain carrying a targeted deletion of ndk, the structural gene for nucleoside diphosphate kinase. How do bacteria lacking this essential housekeeping enzyme synthesize nucleoside triphosphates? In view of the specific interactions of nucleoside diphosphate kinase with T4 enzymes of DNA metabolism, how does T4 multiply after infection of this host? Finally, the ndk disruption strain has highly biased nucleoside triphosphate pools, including elevations of the CTP and dCTP pools of 7- and 23-fold, respectively. Accompanied by these biased nucleotide pools is a strong mutator phenotype. What is the biochemical basis for the pool abnormalities and what are the mutagenic mechanisms? We conclude with brief references to related work in other laboratories.

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...

We have used 8-azidoadenosine 5-triphosphate (8-N 3 ATP) to investigate the nucleotide-binding sites on the NrdD subunit of the anaerobic ribonucleotide reductase from T4 phage. Saturation studies revealed two saturable sites for this photoaffinity analog of ATP. One site exhibited half-maximal saturation at approximately 5 M [␥-32 P]8-N 3 ATP, whereas the other site required 45 M. To localize the sites of photoinsertion, photolabeled peptides from tryptic and chymotryptic digests were isolated by immobilized Al 3؉ affinity chromatography and high performance liquid chromatography and subjected to amino acid sequence and mass spectrometric analyses. The molecular masses of the photolabeled products of cyanogen bromide cleavage were estimated using tricine-SDS-polyacrylamide gel electrophoresis. Overlapping sequence analysis localized the higher affinity site to the region corresponding to residues 289 -291 and the other site to the region corresponding to residues 147-160. Site-directed mutagenesis of Cys 290 , a residue conserved in all known class III reductases, resulted in a protein that exhibited less than 10% of wild type enzymatic activity. These observations indicate that Cys 290 may reside in or near the active site. High performance liquid chromatography analysis revealed that photoinsertion of [␥-32 P]8-N 3 ATP into the site corresponding to residues 147-160 was almost completely abolished when 100 M dATP, dGTP, or dTTP was included in the photolabeling reaction mixture, whereas 100 M ATP, GTP, CTP, or dCTP had virtually no effect. Based on these nucleotide binding properties, we conclude that this site is an allosteric site analogous to the one that has been shown to regulate substrate specificity of other ribonucleotide reductases. There was no evidence for a second allosteric nucleotide-binding site as observed in the anaerobic ribonucleotide reductase from Escherichia coli.Ribonucleotide reductases catalyze the reduction of ribonucleotides to their corresponding 2Ј-deoxyribonucleotides. Currently, they are divided into three classes based on differences in cofactor requirements, structural composition, and type of radical employed for catalysis (1, 2). Because of the importance of maintaining a balanced supply of deoxyribonucleotides for DNA synthesis (3, 4), they are enzymes that are subject to complex allosteric regulation (5, 6). Although there may be striking differences in primary sequence, the same general mechanism for allosteric regulation appears to apply to all ribonucleotide reductases described to date, with only subtle differences observed (7). Three different nucleotide-binding sites have been localized on the prototypical class I reductase from Escherichia coli using photoaffinity labeling (8) and x-ray crystallography (9, 10). Two of these sites are allosteric sites that coordinate the reduction of all four ribonucleotide substrates at a single active site. One allosteric site binds only ATP and dATP and regulates the overall activity of the enzyme. The other allosteric site, via i...

Using 'ZP-labeled 2-azidoadenosine 5'-triphosphate (2N,ATP) and 8-azidoadenosine 5'-triphosphate (8N,ATP), we have identified a site on human interferon a2 (IFN-a2) that binds adenine nucleotides. The results from saturation and competition expeiinients demonstrated the specificity of the nucleotide interaction. Half-maximal saturation of IFN-a2 was observed at 10 pM 2N,ATP or 35 pM 8N,ATP. ATP effectively decreased photoinsertion of both photoaffinity analogs of ATP. Photoinsertion of 8N,ATP was enhanced by MgCI,, independent of the ionic strength, and exhibited an optimum pH between 7.0 and 7.5. Immobilized-Al" affinity chromatography and HPLC were used to purify the modified peptides from IFN-a2 that had been photolabeled with 8N,ATP and digested with trypsin or chymotrypsin. Overlappingsequence analysis localized the sites of photoinsertion to the region corresponding to Lysl21 -Tyr135 in the amino acid sequence of IFN-a2, which almost perfectly overlaps a nuclear-localization signal (R120KYFQRITLYLKEKKY 135).Keywords: interferon a ; photoaffinity labeling ; nucleotide binding ; nuclear localization signal ; ATP. While most research has focused on the receptor's role in the generation of second messengers, an accumulating amount of evidence suggests that there may be an intracellular role for the interferons. Early studies revealed that colchicine and cytochalasin, two agents known to affect internalization of various polypeptide hormones, impair the antiviral activity of interferon