Succinyl coenzyme A synthetase of Pseudomonas aeruginosa with a broad specificity for nucleoside triphosphate (NTP) synthesis modulates specificity for NTP synthesis by the 12-kilodalton form of nucleoside diphosphate kinase

Pseudomonas aeruginosa secretes copious amounts of an exopolysaccharide called alginate during infection in the lungs of cystic fibrosis patients. A mutation in the algR2 gene of mucoid P. aeruginosa is known to exhibit a nonmucoid (nonalginate-producing) phenotype and showed reduced activities of succinyl-coenzyme A (CoA) synthetase (Scs) and nucleoside diphosphate kinase (Ndk), implying coregulation of Ndk and Scs in alginate synthesis. We have cloned and characterized the sucCD operon encoding the alpha and beta subunits of Scs from P. aeruginosa and have studied the role of Scs in generating GTP, an important precursor in alginate synthesis. We demonstrate that, in the presence of GDP, Scs synthesizes GTP using ATP as the phosphodonor and, in the presence of ADP, Scs synthesizes ATP using GTP as a phosphodonor. In the presence of inorganic orthophosphate, succinyl-CoA, and an equimolar amount of ADP and GDP, Scs synthesizes essentially an equimolar amount of ATP and GTP. Such a mechanism of GTP synthesis can be an alternate source for the synthesis of alginate as well as for the synthesis of other macromolecules requiring GTP such as RNA and protein. Scs from P. aeruginosa is also shown to exhibit a broad NDP kinase activity. In the presence of inorganic orthophosphate (P(i)), succinyl-CoA, and either GDP, ADP, UDP or CDP, it synthesizes GTP, ATP, UTP, or CTP. Scs was previously shown to copurify with Ndk, presumably as a complex. In mucoid cells of P. aeruginosa, Ndk is also known to exist in two forms, a 16-kDa cytoplasmic form predominant in the log phase and a 12-kDa membrane-associated form predominant in the stationary phase. We have observed that the 16-kDa Ndk-Scs complex present in nonmucoid cells, synthesizes all three of the nucleoside triphosphates from a mixture of GDP, UDP, and CDP, whereas the 12-kDa Ndk-Scs complex specifically present in mucoid cell predominantly synthesizes GTP and UTP but not CTP. Such regulation may promote GTP synthesis in the stationary phase when the bulk of alginate is synthesized by mucoid P. aeruginosa.     

Conversion of GDP into GTP by nucleoside diphosphate kinase on the GTP-binding proteins

A direct interaction of alpha beta gamma trimeric GTP binding proteins (G proteins; G0 and Gs) with nucleoside diphosphate kinase (NDP kinase) was investigated with homogeneously purified proteins. There was a progressive release of 32Pi from [gamma-32P]ATP when GDP-bound G0 was incubated together with NDP kinase. The Pi release induced by the interaction of G0 with NDP kinase was not accompanied by the dissociation of GDP bound to the alpha-subunit of G0. This was a sharp contrast to G protein-catalyzed GTP hydrolysis observed with GTP as the substrate; the dissociation of bound GDP was essentially required for the following binding of the substrate, GTP, to be hydrolyzed. A kinetic analysis displayed different properties for the substrate of NDP kinase between free GDP and G protein-bound GDP. NDP kinase-dependent phosphorylation of GDP on G0 was indeed demonstrated with adenosine 5'-(3-O-thio)triphosphate as the phosphate donor; there was a formation of guanosine 5'-(3-O-thio)triphosphate-bound G0 from the ATP analogue. Moreover, purified Gs was readily ADP-ribosylated by cholera toxin in the presence of NDP kinase, ATP, and an ADP-ribosylation factor, also suggesting that the nucleotide form on Gs was certainly GTP. These results indicate that NDP kinase can transfer the gamma-phosphate of ATP directly to GDP bound to G proteins and that this phosphorylation results in the activation of the signal-coupling proteins. A possible role of the new activation mechanism of G proteins is discussed in comparison with the previously characterized GDP-GTP exchange pathway by the agonist-receptor complex.     

Cytotoxic activity of nucleoside diphosphate kinase secreted from Mycobacterium tuberculosis.

Pathogenicity of Mycobacterium tuberculosis is closely related to its ability to survive and replicate in the hostile environment of macrophages. For some pathogenic bacteria, secretion of ATP-utilizing enzymes into the extracellular environment aids in pathogen survival via P2Z receptor-mediated, ATP-induced death of infected macrophages. A component of these enzymes is nucleoside diphosphate kinase (Ndk). The ndk gene was cloned from M. tuberculosis H37Rv and expressed in Escherichia coli. Ndk was secreted into the culture medium by M. tuberculosis, as determined by enzymatic activity and Western blotting. Purified Ndk enhanced ATP-induced macrophage cell death, as assayed by the release of [14C]adenine. A catalytic mutant of Ndk failed to enhance ATP-induced macrophage cell death, and periodate-oxidized ATP (oATP), an irreversible inhibitor of P2Z receptor, blocked ATP/Ndk-induced cell death. Purified Ndk was also found to be autophosphorylated with broad specificity for all nucleotides. Conversion of His117-->Gln, which is part of the nucleotide-binding site, abolished autophosphorylation. Purified Ndk also showed GTPase activity. Collectively, these results indicate that secreted Ndk of M. tuberculosis acts as a cytotoxic factor for macrophages, which may help in dissemination of the bacilli and evasion of the immune system.     

Regulation of dynamin by nucleoside diphosphate kinase

Nucleoside diphosphate (NDP) kinase is required for multiple cellular functions, including cell growth, motility, and differentiation, and its loss is associated with pathologies including tumor metastasis. A recent study has revealed a previously unknown function for NDP kinase as positive regulator of dynamin, a GTPase essential for endocytosis. In this review we describe the evidence that NDP kinase function is essential for endocytosis and also elaborate on a mechanism for NDP kinase regulation of dynamin. Recently documented interactions between endocytosis and cell signaling have revealed new insights into potential mechanisms of cancer. In this context, we discuss the possible relevance of NDP kinase and dynamin interaction for tumor suppression.     

Phosphorylation of anti-HIV nucleoside analogs by nucleoside diphosphate kinase.

The reaction of NDP kinase with antiviral nucleoside triphosphates used in antiviral therapies was studied at the presteady state by fluorescence stopped-flow and compared with the steady-state parameters. The affinity of the analogs was determined by fluorescence titration of a mutated enzyme with an inserted Trp in the binding site. The lack of the 3' hydroxyl in analogs is shown to decrease the kcat more than the KD.     

Regulation of nucleoside diphosphate kinase and an alternative kinase in Escherichia coli: role of the sspA and rnk genes in nucleoside triphosphate formation

We have previously reported that two genes cloned from a cosmid library of Escherichia coli can restore mucoidy to an algR2 mutant of Pseudomonas aeruginosa . AlgR2 is a protein involved in the regulation of nucleoside diphosphate kinase (Ndk) as well as alginate synthesis in P. aeruginosa . One of the E. coli genes, rnk , encodes a 14.9 kDa protein with no homology to any other proteins. The other gene, sspA , encodes the stringent starvation protein, a regulatory protein involved in stationary-phase regulation and the stringent response of E. coli . While both rnk and sspA restored alginate production to the P. aeruginosa algR2 mutant, only rnk restored Ndk activity to the mutant. In this report, we have examined the effect of mutations in rnk and sspA on the levels of Ndk in E. coli . We find that a mutation in rnk drastically reduces the level of Ndk in E. coli . A mutation in sspA , however, affects the level of another nucleoside diphosphate kinase distinct from Ndk. The proteins can be easily distinguished from each other by their different affinities for nucleoside diphosphates (NDPs) and also by the differential effect of anti-Ndk antibodies on the reactions they catalyse. The ability of either of these two proteins to restore alginate synthesis in the algR2 mutant of P. aeruginosa demonstrates the importance of nucleoside triphosphate synthesis and energy metabolism for alginate synthesis. Additionally, a role for the stringent starvation protein (SspA) in the modulation of nucleoside triphosphate (NTP) levels in E. coli is also suggested from these experiments.     

Cytosolic adenylate kinase catalyzes the synthesis of thiamin triphosphate from thiamin diphosphate

An attempt was made to purify a porcine skeletal muscle enzyme catalyzing the formation of thiamin triphosphate (TTP) from thiamin diphosphate (TDP), requiring ATP, Mg2+ and a cofactor (creatine). As the purification proceeded, the reaction requirements for ATP and creatine were lost and then a requirement for ADP was manifested. The activity responsible for TTP synthesis from TDP, ADP, and Mg2+ was found to be copurified with adenylate kinase [EC 2.7.4.3] activity, and was finally purified to a single band on SDS-PAGE. Antiserum obtained against the purified enzyme preparation inhibited both adenylate kinase activity and the TTP-synthesizing activity to exactly the same extent. These results indicate that adenylate kinase catalyzes TTP formation from TDP in vitro.     

Characterization of membrane-associated Pseudomonas aeruginosa Ras-like protein Pra, a GTP-binding protein that forms complexes with truncated nucleoside diphosphate kinase and pyruvate kinase to modulate GTP synthesis.

We report the purification and characterization of a protein from the membrane fraction of Pseudomonas aeruginosa showing intrinsic guanosine triphosphatase (GTPase) activity. The protein was purified as a 48-kDa polypeptide capable of binding and hydrolyzing GTP. The N-terminal sequence of the purified protein revealed its similarity to the Escherichia coli Ras-like protein (Era), and the protein cross-reacted with anti-Era antibodies. This protein was named Pseudomonas Ras-like protein (Pra). Anti-Pra antibodies also cross-reacted with E. coli Era protein. Pra is autophosphorylated in vitro, with phosphotransfer of the terminal phosphate from [gamma-32P]GTP but not [gamma-32P]ATP. Pra is capable of complex formation with the truncated 12-kDa form of nucleoside diphosphate kinase (Ndk) but not with the 16-kDa form. Purified Pra was also shown to physically interact with pyruvate kinase (Pk); Pk and Pra can form a complex, but when the 12-kDa Ndk, Pk, and Pra are all present, Pk has a higher affinity than Pra for forming a complex with the 12-kDa Ndk. The 12-kDa Ndk-Pra complex catalyzed increased synthesis of GTP and dGTP and diminished synthesis of CTP and UTP or dCTP and dTTP relative to their synthesis by uncomplexed Ndk. Moreover, the complex of Pra with Pk resulted in the specific synthesis of GTP as well when Pra was present in concentrations in excess of that of Pk. Membrane fractions from cells harvested in the mid-log phase demonstrated very little nucleoside triphosphate (NTP)-synthesizing activity and no detectable Ndk. Membranes from cells harvested at late exponential phase showed NTP-synthesizing activity and the physical presence of Ndk but not of Pk or Pra. In contrast, membrane fractions of cells harvested at early to late stationary phase showed predominant GTP synthesis and the presence of increasing amounts of Pk and Pra. It is likely that the association of Pra with Ndk and/or Pk restricts its intrinsic GTPase activity, which may modulate stationary-phase gene expression and the survival of P. aeruginosa by modulating the level of GTP.     

Escherichia coli nucleoside diphosphate kinase interactions with T4 phage proteins of deoxyribonucleotide synthesis and possible regulatory functions

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.     

Isolation of a mRNA encoding a nucleoside diphosphate kinase from tomato that is up-regulated by wounding

A cDNA clone (TAB2) encoding a nucleoside diphosphate (NDP) kinase has been isolated from a tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) cDNA library. The clone is 590 bp long and exhibits a high degree of sequence identity with spinach NDP kinases I and II, Pisum sativum NDP kinase I, Arabidopsis thaliana NDP kinase, Drosophila melanogaster NDP kinase, Dictyostelium discoideum NDP kinase and human Nm 23-H1 and Nm23-H2. Northern analysis has revealed that the mRNA encoded by TAB2 is up-regulated in both leaf and stem tissue in response to wounding. The increase is apparent within 1 h of wounding and is not further elevated by application of ethylene. Southern blot analysis indicates that TAB2 is a member of a small gene family.     

The structure of the Escherichia coli nucleoside diphosphate kinase reveals a new quaternary architecture for this enzyme family

Nucleoside diphosphate kinase (NDPK) catalyzes the transfer of gamma-phosphate from nucleoside triphosphates to nucleoside diphosphates. The subunit folding and the dimeric basic structural unit are remarkably the same for available structures but, depending on species, dimers self-associate to form hexamers or tetramers. The crystal structure of the Escherichia coli NDPK reveals a new tetrameric quaternary structure for this protein family. The two tetramers differ by the relative orientation of interacting dimers, which face either the convex or the concave side of their central sheet as in either Myxococcus xanthus (type I) or E. coli (type II), respectively. In the type II tetramer, the subunits interact by a new interface harboring a zone called the Kpn loop as in hexamers, but by the opposite face of this loop. The evolutionary conservation of the interface residues indicates that this new quaternary structure seems to be the most frequent assembly mode in bacterial tetrameric NDP kinases.     

NMR studies on the flexibility of nucleoside diphosphate kinase

Human NDP kinase B, product of the nm23-H2 gene, binds DNA. It has been suggested that a helix hairpin on the protein surface, part of the nucleotide substrate binding site, could accommodate DNA binding by swinging away. The presence of flexible regions was therefore investigated by ¹H NMR dynamic filtering. Although TOCSY peaks could be assigned to five residues at the N terminus of Dictyostelium NDP kinase, no flexible region was detected in the human enzyme. These data favor the idea that the protein offers different binding sites to mono- and polynucleotides. Proteins 28:150–152, 1997. © 1997 Wiley-Liss Inc.     

Evidence that the isomerization of D-ribose and L-rhamnose is catalyzed by the same enzyme in Mycobacterium smegmatis.

D-Ribose isomerase was purified and crystallized from cells of Mycobacterium smegmatis grown on either D-ribose or L-rhamnose. Isomerase activity for both of these sugars remained together throughout the purification. The isomerase from L-rhamnose-grown cells had the same chemical and physical properties as the enzyme isolated from D-ribose grown cells. In addition, immunological studies indicated that both activities were in the same protein since antisera prepared against either of the crystals cross-reacted with the other and gave lines of symmetry by the agar gel diffusion method.