The microtubule-associated nucleoside diphosphate kinase

Microtubule protein prepared by cycles of assembly-disassembly contains a nucleoside diphosphate kinase (NDP kinase) activity. We have isolated the NDP kinase responsible for this activity from twice-polymerized bovine brain microtubule protein by a five-step chromatographic procedure. The molecular weight of this enzyme was 103,000 +/- 7,000 daltons as determined by sedimentation equilibrium experiments performed with a Beckman Airfuge. A doublet of subunit bands with molecular masses of about 18,000 daltons was detected by silver staining after gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this preparation. We conclude that the enzyme is a hexamer, although we cannot identify the mix of subunits. We were able to isolate only nanogram quantities of this enzyme, too little for extensive studies, so we isolated the enzyme directly from bovine brain without a preliminary microtubule protein isolation. The whole-brain NDP kinase was isolated by the same chromatographic steps as the enzyme from microtubule protein preparations. Both enzymes had a doublet of subunits at the same molecular weights and both were the same isozyme, chromatofocusing at a pH of 8.0. Both enzymes had similar kinetic properties and similar thermal inactivation profiles. These similar properties of the two enzymes suggest that they are identical. Both subunits of NDP kinase could be reversibly phosphorylated by ATP. Phosphorylation of the native enzyme created multiple, more acidic forms that retained activity. The isolation of this NDP kinase, which can copurify with microtubule protein through cycles of assembly-disassembly, will facilitate future studies on the role of this enzyme in the mechanism and regulation of microtubule assembly.     

Interaction of hexa-His tag with acidic amino acids results in facilitated refolding of halophilic nucleoside diphosphate kinase

We have previously reported that amino-terminal extension sequence containing hexa-His facilitated refolding and assembly of hexameric nucleoside diphosphate kinase from extremely halophilic archaeon Halobacterium salinarum (NDK). In this study, we made various mutations in both the tag sequence and within NDK molecule. SerNDK, in which hexa-His was replaced with hexa-Ser, showed no facilitated folding. In addition, HisD58GD63G, in which both Asp58 and Asp63 in NDK were replaced with Gly, also showed no refolding enhancement. These results suggest that hexa-His in His-tag interact cooperatively with either Asp58 or Asp63 or both. Furthermore, G114D mutant, which formed a dimer in low salt solution, was strongly stabilized by His-tag to form a stable hexamer.     

Import and localisation of nucleoside diphosphate kinase 2 in chloroplasts

Nucleoside diphosphate kinases (NDPKs) are key enzymes that are involved in the homeostasis of nucleoside triphosphates (NTPs). Different isoforms exist, which are found in diverse cell compartments, for example the cytosol, mitochondria, and plant chloroplasts. NDPK2 of Pisum sativum has been shown to be localised in chloroplasts. Two forms of different size have been reported in plastids and it has been speculated that they function in distinct suborganellar compartments. We investigated the import behaviour and localisation of these two isoforms. Our results indicate that they do not differ in their route of entry into the organelle and both forms end up in the chloroplast stroma.     

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.     

X-ray structure of nucleoside diphosphate kinase.

The X-ray structure of a point mutant of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been determined to 2.2 A resolution. The enzyme is a hexamer made of identical subunits with a novel mononucleotide binding fold. Each subunit contains an alpha/beta domain with a four stranded, antiparallel beta-sheet. The topology is different from adenylate kinase, but identical to the allosteric domain of Escherichia coli ATCase regulatory subunits, which bind mononucleotides at an equivalent position. Dimer contacts between NDP kinase subunits within the hexamer are similar to those in ATCase. Trimer contacts involve a large loop of polypeptide chain that bears the site of the Pro----Ser substitution in Killer of prune (K-pn) mutants of the highly homologous Drosophila enzyme. Properties of Drosophila NDP kinase, the product of the awd developmental gene, and of the human enzyme, the product of the nm23 genes in tumorigenesis, are discussed in view of the three-dimensional structure and of possible interactions of NDP kinase with other nucleotide binding proteins.     

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.     

Post-translational processing of Drosophila nucleoside diphosphate kinase

Nucleoside diphosphate kinase (NDPK) was purified from Drosophila melanogaster by a combination of anion-exchange, hydroxyapatite, and reversed-phase chromatography. The identity of the purified enzyme was confirmed by sequencing internal peptides (the N-terminus appeared to be blocked). Post-translational modifications were investigated by using protein chemical and mass spectrometric methods. Analysis by nanoelectrospray ionization-mass spectrometry revealed that the mass of the enzyme was considerably smaller than that predicted from its amino acid sequence. Although its open-reading frame predicts a 153-residue polypeptide, the mature enzyme was found to comprise 152 amino acids, being modified by proteolytic removal of the initiator Met and N-acetylation of Ala2. This explains why the observed pI of the Drosophila enzyme is more acidic than that predicted from its amino acid sequence. No additional post-translational modifications such as glycosylation or O-phosphorylation, which have been identified on homologous NDPKs from other organisms, were detected on the Drosophila enzyme.     

Polyphosphate kinase 2: a modulator of nucleoside diphosphate kinase activity in mycobacteria

Mycobacteria encode putative class II polyphosphate kinases (PPKs). We report that recombinant PPK2 of Mycobacterium tuberculosis catalyses the synthesis of GTP from GDP using polyphosphate rather than ATP as phosphate donor. Unlike that of PPK1, this is the favoured reaction of PPK2. The sites of autophosphorylation, H115 and H247, as well as G74 were critical for GTP‐synthesizing activity. Compromised survival of a ppk2 knockout (PPK2‐KO) of Mycobacterium smegmatis under heat or acid stress or hypoxia, and the ability of ppk2 of M. tuberculosis to complement this, confirmed that PPK2 plays a role in mycobacterial survival under stress. Intracellular ATP : GTP ratio was higher in PPK2‐KO compared with the wild‐type M. smegmatis, bringing to light a role of PPK2 in regulating the intracellular nucleotide pool. We present evidence that PPK2 does so by interacting with nucleoside diphosphate kinase (Ndk). Pull‐down assays and analysis by surface plasmon resonance demonstrated that the interaction requires G74 of PPK2_MTB and ¹⁰⁹LET¹¹¹ of Ndk_MTB. In summary, we unravel a novel mechanism of regulation of nucleotide pools in mycobacteria. Downregulation of ppk2 impairs survival of M. tuberculosis in macrophages, suggesting that PPK2 plays an important role in the physiology of the bacteria residing within macrophages.     

Biochemical characteristics of bovine retina nucleoside diphosphate kinase

Nucleoside diphosphate kinase (NDP kinase; ATP: NDP phosphotransferase; EC 2.7.4.6) was purified from bovine retina. The molecular mass of the native enzyme was found to be 72 kDa, and those of its subunits were 17.5 and 18.5 kDa. Kinetic characteristics of the enzyme were determined. It was shown that NDP kinase exists in retina in both soluble and membrane-bound forms.     

Molecular interactions involving Escherichia coli nucleoside diphosphate kinase

Nucleoside diphosphate kinase plays a distinctive metabolic role as the enzyme poised between the last reaction of deoxyribonucleoside triphosphate (dNTP) biosynthesis and the DNA polymerization apparatus. In bacteriophage T4 infection, NDP kinase is one of very few enzymes of host cell origin to participate in either dNTP synthesis or DNA replication. Yet NDP kinase forms specific contacts with phage-coded proteins of dNTP and DNA synthesis. This article summarizes work from our laboratory that identifies and characterizes these interactions. Despite these specific interactions, the enzyme appears to be dispensable, both for T4 replication and for growth of the host, Escherichia coli, because site-specific disruption of ndk, the structural gene for NDP kinase, does not interfere with growth of the host cell and only partly inhibits phage replication. However, ndk disruption unbalances the dNTP pools and stimulates mutagenesis. We discuss our attempts to understand the basis for this enhanced mutagenesis.     

Effects of mutations at Gly114 on the stability and refolding of haloarchaeal nucleoside diphosphate kinase in low salt solution

We have shown before that mutation of Gly114 to Arg enhances folding of hexameric nucleoside diphosphate kinase (HsNDK) from Halobacterium salinarum. In this study, we constructed three mutant forms, Gly114Lys (G114K), Gly114Ser (G114S) and Gly114Asp (G114D), to further clarify the role residue 114 plays in the stability and folding of HsNDK. While expression of G114D mutant resulted in inactive enzyme, other mutant HsNDKs were successfully expressed in active form. The G114K mutant, similar to Gly114Arg (G114R) mutant, refolded in 1 M NaCl after heat-denaturation, under which the wild-type HsNDK and G114S proteins showed no refolding.     

Flagellar radial spokes contain a Ca2+-stimulated nucleoside diphosphate kinase

The radial spokes are required for Ca(2+)-initiated intraflagellar signaling, resulting in modulation of inner and outer arm dynein activity. However, the mechanochemical properties of this signaling pathway remain unknown. Here, we describe a novel nucleoside diphosphate kinase (NDK) from the Chlamydomonas flagellum. This protein (termed p61 or RSP23) consists of an N-terminal catalytic NDK domain followed by a repetitive region that includes three IQ motifs and a highly acidic C-terminal segment. We find that p61 is missing in axonemes derived from the mutants pf14 (lacks radial spokes) and pf24 (lacks the spoke head and several stalk components) but not in those from pf17 (lacking only the spoke head). The p61 protein can be extracted from oda1 (lacks outer dynein arms) and pf17 axonemes with 0.5 M KI, and copurifies with radial spokes in sucrose density gradients. Furthermore, p61 contains two classes of calmodulin binding site: IQ1 interacts with calmodulin-Sepharose beads in a Ca(2+)-independent manner, whereas IQ2 and IQ3 show Ca(2+)-sensitive associations. Wild-type axonemes exhibit two distinct NDKase activities, at least one of which is stimulated by Ca(2+). This Ca(2+)-responsive enzyme, which accounts for approximately 45% of total axonemal NDKase, is missing from pf14 axonemes. We found that purified radial spokes also exhibit NDKase activity. Thus, we conclude that p61 is an integral component of the radial spoke stalk that binds calmodulin and exhibits Ca(2+)-controlled NDKase activity. These observations suggest that nucleotides other than ATP may play an important role in the signal transduction pathway that underlies the regulatory mechanism defined by the radial spokes.     

Trypanosoma cruzi nucleoside diphosphate kinase 1 ( TcNDPK1) has a broad nuclease activity

Here, we present the characterization of a trypanosomatid nucleoside diphosphate kinase (TcNDPK1) exhibiting nuclease activity. This is the first identification of a NDPK with this property in trypanosomatid organisms. The recombinant TcNDPK1 protein cleaves not only linear DNA, but also supercoiled plasmid DNA. Additionally, TcNDPK1 is capable of degrading Trypanosoma cruzi genomic DNA. ATP or ADP did not affect the nuclease activity, while the absence of Mg2+ completely inhibits this activity. NDPK and nuclease activities were inhibited at the same temperature, suggesting the presence of related catalytic sites. Furthermore, phenogram analysis showed that TcNDPK1 is close to Drosophila melanogaster and human NDPKs. The unspecific nuclease activity could suggest a participation in cellular processes such as programmed cell death.     

Distribution and properties of human liver nucleoside diphosphate kinase

• 1.1. Human liver nucleoside diphosphate kinase is predominantly cytosolic (64%). A small fraction (9.2%) is located into the mitochondria, but an unexpectedly high activity (21%) was found in microsomes.
• 2.2. Like other sources of nucleoside diphosphate kinase, the human enzyme has a broad specificity towards nucleotides both as donors or acceptors.
• 3.3. Serum nucleoside diphosphate kinase activity in the presence of ATP and 8-bromoIDP emphasized mean values of 15 IU/1. No significant increase of serum nucleoside diphosphate kinase activity was noted in patients with acute viral hepatitis.

     

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.