Nucleoside diphosphate kinase from Xenopus oocytes; partial purification and characterization

We have identified and partially purified a soluble nucleoside diphosphate kinase (NDP kinase) from Xenopus laevis oocytes. The enzyme preparation can catalyze the transfer of phosphate from ATP to all of the major oxy- and deoxynucleotides. It can also catalyze the transfer of a phosphorothioate group from gamma-S-ATP to an acceptor GDP forming gamma-S-GTP. Like NDP kinases from other sources, the catalytic mechanism appears to involve a phosphoenzyme intermediate which can be isolated. Transfer of phosphate from nucleoside triphosphates to protein is rapid, reaching saturation within 1 min following the addition of nucleoside triphosphates. The transfer of phosphate from phosphoprotein intermediate to nucleoside diphosphates is equally fast. While nucleoside diphosphate kinases are generally thought to require magnesium for activity, both the oocyte enzyme preparation and a commercial bovine liver enzyme preparation are only partially inhibited by short (10 min) exposures to 25 mM EDTA. Both enzyme preparations are, however, further inhibited by long incubations with this metal chelator (2 h, 70% inhibition). Zinc enhances the inhibition of NDP kinase by EDTA, but is ineffective on its own. Rapid phosphorylation in the presence of [gamma-32P]ATP and EDTA could be used to identify the phosphoenzyme intermediate in homogenates of Xenopus oocytes and facilitated its isolation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with autoradiography indicated the presence of only a single phosphorylated species of Mr 21,500 in supernatants of fresh oocyte homogenates. Partial purification of this protein utilizing salt precipitation, hydrophobic-interaction chromatography and an affinity step with Affi-Gel Blue Sepharose resulted in a 100-fold purification and a 29% overall yield of NDP-kinase activity. Size-exclusion chromatography of the purified preparation yielded two peaks containing enzyme activity. They eluted with apparent molecular weights of 45,000 and 70,000, suggesting a native enzyme that is multimeric or associated with other proteins.     

The Human Nm23/Nucleoside Diphosphate Kinases

Biochemical experiments over the past 40 years have shown that nucleoside diphosphate(NDP) kinase activity, which catalyzes phosphoryl transfer from a nucleoside triphosphate toa nucleoside diphosphate, is ubiquitously found in organisms from bacteria to human. Overthe past 10 years, eight human genes of the nm23/NDP kinase family have been discoveredthat can be separated into two groups based on analysis of their sequences. In addition tocatalysis, which may not be exhibited by all isoforms, evidence for regulatory roles has comerecently from the discovery of the genes nm23 and awd, which encode NDP kinases and areinvolved in tumor metastasis and Drosophila development, respectively. Current work showsthat the human NDP kinase genes are differentially expressed in tissues and that their productsare targeted to different subcellular locations. This suggests that Nm23/NDP kinases possessdifferent, but specific, functions within the cell, depending on their localization. The roles ofNDP kinases in metabolic pathways and nucleic acid synthesis are discussed.     

Putative functions of nucleoside diphosphate kinase in plants and fungi

The putative functions of NDP (nucleoside diaphosphate) kinases from various organisms focusing to fungi and plants are described. The biochemical reactions catalyzed by NDP kinase are as follows. (i) Phosphotransferring activity from mainly ATP to cognate NDPs generating nucleoside triphosphates (NTPs). (ii) Autophosphorylation activity from ATP and GTP. (iii) Protein kinase (phosphotransferring) activity phosphorylating such as myelin basic protein. NDP kinase could function to provide NTPs as a housekeeping enzyme. However, recent works proved possible functions of the NDP kinases in the processes of signal transduction in various organisms, as described below. By use of the extracts of the mycelia of a filamentous fungus Neurospora crassa blue-light irradiation could increase the phosphorylation of a 15-kDa protein, which was purified and identified to be NDP kinase (NDK-1). By use of the etiolated seedlings of Pisum sativum cv Alaska and Oryza sativa red-light irradiation of intact plants increased the phosphorylation of NDP kinase. However, successive irradiation by red–far-red reversed the reaction, indicating that phytochrome-mediated light signals are transduced to the phosphorylation of NDP kinase. NDP kinase localizing in mitochondria is encoded by nuclear genome and different from those localized in cytoplasm. NDP kinase in mitochondria formed a complex with succinyl CoA synthetase. In Spinicia oleraceae two different NDP kinases were detected in the chloroplast, and in Pisum sativum two forms of NDP kinase originated from single species of mRNA could be detected in the choloroplast. However, the function of NDP kinases in the choloroplast is not yet known. In Neurospora crassa a Pro72His mutation in NDP kinase (ndk-1 ^Pro72His ) deficient in the autophosphorylation and protein kinase activity resulted in lacking the light-induced polarity of perithecia. In wild-type directional light irradiation parallel to the solid medium resulted in the formation of the perithecial beak at the top of perithecia, which was designated as light-induced polarity of perithecia. In wild-type in darkness the beak was formed at random places on perithecia, and in ndk ^Pro72His mutant the perithecial beak was formed at random places even under directional light illumination. The introduction of genomic DNA and cDNA for ndk-1 demonstrated that the wild-type DNAs suppressed the mutant phenotype. With all these results except for the demonstration in Neurospora, most of the phenomena are elusive and should be solved in the molecular levels concerning with NDP kinases.     

Autophosphorylation of nucleoside diphosphate kinase from Myxococcus xanthus.

The nucleoside diphosphate kinase (NDP kinase) from Myxococcus xanthus has been purified to homogeneity and crystallized (J. Munoz-Dorado, M. Inouye, and S. Inouye, J. Biol. Chem. 265:2702-2706, 1990). In the presence of ATP, the NDP kinase was autophosphorylated. Phosphoamino acid analysis was carried out after acid and base hydrolyses of phosphorylated NDP kinase. It was found that the protein was phosphorylated not only at a histidine residue but also at a serine residue. Replacement of histidine 117 with a glutamine residue completely abolished the autophosphorylation and nucleotide-binding activity of the NDP kinase. Since histidine 117 is the only histidine residue that is conserved in all known NDP kinases so far characterized, the results suggest that the phosphohistidine intermediate is formed at this residue during the transphosphorylation reaction from nucleoside triphosphates to nucleoside diphosphates. Preliminary mutational analysis of putative ATP-binding sites is also presented.     

Nucleotide binding to nucleoside diphosphate kinases: X-ray structure of human NDPK-A in complex with ADP and comparison to protein kinases

NDPK-A, product of the nm23-H1 gene, is one of the two major isoforms of human nucleoside diphosphate kinase. We analyzed the binding of its nucleotide substrates by fluorometric methods. The binding of nucleoside triphosphate (NTP) substrates was detected by following changes of the intrinsic fluorescence of the H118G/F60W variant, a mutant protein engineered for that purpose. Nucleoside diphosphate (NDP) substrate binding was measured by competition with a fluorescent derivative of ADP, following the fluorescence anisotropy of the derivative. We also determined an X-ray structure at 2.0A resolution of the variant NDPK-A in complex with ADP, Ca(2+) and inorganic phosphate, products of ATP hydrolysis. We compared the conformation of the bound nucleotide seen in this complex and the interactions it makes with the protein, with those of the nucleotide substrates, substrate analogues or inhibitors present in other NDP kinase structures. We also compared NDP kinase-bound nucleotides to ATP bound to protein kinases, and showed that the nucleoside monophosphate moieties have nearly identical conformations in spite of the very different protein environments. However, the beta and gamma-phosphate groups are differently positioned and oriented in the two types of kinases, and they bind metal ions with opposite chiralities. Thus, it should be possible to design nucleotide analogues that are good substrates of one type of kinase, and poor substrates or inhibitors of the other kind.     

Real-time bioluminometric method for detection of nucleoside diphosphate kinase activity.

A real-time, simple and sensitive method for detection of nucleoside diphosphate (NDP) kinase activity has been developed. The assay is based on detection of ATP, generated in the NDP kinase reaction between a nucleoside triphosphate and adenosine diphosphate (ADP), by the firefly luciferase system. In the presence of 0.3 mM dGTP, the Km for ADP was found to be approximately 30 microM for the NDP kinase from Baker's yeast. In the presence of 250 microM ADP, the Km for dATP alpha S, dTTP alpha S, dGTP, dTTP, dCTP and GTP was found to be approximately 0.01, 0.03, 0.05, 0.25, 0.75 and 0.2 mM, respectively. The assay is sensitive and yields linear responses between 0.05-50 mU. The detection limit was found to be 0.05 mU of NDP kinase. The method was used to detect NDP kinase contamination in commercial enzyme preparations.     

Structural and catalytic properties and homology modelling of the human nucleoside diphosphate kinase C, product of the DRnm23 gene.

The human DRnm23 gene was identified by differential screening of a cDNA library obtained from chronic myeloid leukaemia-blast crisis primary cells. The over-expression of this gene inhibits differentiation and induces the apoptosis of myeloid precursor cell lines. We overproduced in bacteria a truncated form of the encoded protein lacking the first 17 N-terminal amino acids. This truncated protein was called nucleoside diphosphate (NDP) kinase CDelta. NDP kinase CDelta had similar kinetic properties to the major human NDP kinases A and B, but was significantly more stable to denaturation by urea and heat. Analysis of denaturation by urea, using size exclusion chromatography, indicated unfolding without the dissociation of subunits, whereas renaturation occurred via a folded monomer. The stability of the protein depended primarily on subunit interactions. Homology modelling of the structure of NDP kinase CDelta, based on the crystal structure of NDP kinase B, indicated that NDP kinase CDelta had several additional stabilizing interactions. The overall structure of the two enzymes appears to be identical because NDP kinase CDelta readily formed mixed hexamers with NDP kinase A. It is possible that mixed hexamers can be observed in vivo.     

Nucleoside diphosphate kinase from Escherichia coli; its overproduction and sequence comparison with eukaryotic enzymes.

The gene encoding nucleoside diphosphate (NDP) kinase of Escherichia coli was identified by polymerase chain reaction using oligodeoxyribonucleotide primers synthesized on the basis of consensus sequences from Myxococcus xanthus and various eukaryotic NDP kinases. The gene (ndk), mapped at 54.2 min on the E. coli chromosome, was cloned and sequenced. The E. coli NDP kinase was found to consist of 143 amino acid residues that are 57, 45, 45, 42, 43, and 43% identical to the M. xanthus, Dictyostelium discoideum, Drosophila melanogaster, mouse, rat, and human enzymes, respectively. The ndk gene appears to be in a monocistronic operon and, when cloned in a pUC vector, NDP kinase was overproduced at a level of approx. 25% of total cellular proteins. The protein could be labeled with [gamma-32P]ATP and migrated at a 16.5 kDa when electrophoresed in SDS-polyacrylamide gel, which is in good agreement with the Mr of the purified E. coli NDP kinase previously reported.     

Nucleoside diphosphate kinase from brain. Purification and effect on microtubule assembly in vitro

Tubulin strictly requires GTP for its polymerization. Nevertheless, microtubule assembly can be observed in the presence of ATP as the only nucleotide triphosphate, due to the nucleoside diphosphate kinase (NDP kinase) present in microtubule preparations, and which phosphorylates the GDP into GTP. We have purified this enzyme from pig brain to homogeneity, and shown that its relative mass is close to 100 000 in its native state, and 17 000 under denaturing conditions. Therefore it is probably a hexamer, as previously shown for the enzyme from other sources, and also presents a microheterogeneity, with the major isoforms between pI 5.0 and 6.0. The enzyme is transiently phosphorylated during catalysis, as expected within a ping-pong bi-bi mechanism. The effect of the NDP kinase on pure tubulin polymerization was studied: in the presence of NDP kinase, the lag time observed in the kinetics of microtubule assembly was shorter and the final extent of assembly was unchanged. The effect of the enzyme was observed at enzyme concentrations 900-fold lower than tubulin concentration, which shows that the NDP kinase acts catalytically. Kinetic data show that the catalytic effect of the NDP kinase is faster than the rate of nucleotide exchange on tubulin under the same conditions. This result demonstrates that the tubulin-GDP complex itself is a substrate for the enzyme, which may indicate that the GDP bound to tubulin at the E site is exposed on the surface of dimeric tubulin.     

The amino acid sequence of nucleoside diphosphate kinase I from spinach leaves, as deduced from the cDNA sequence.

The primary structure of nucleoside diphosphate (NDP) kinase from spinach leaves has been deduced from its cDNA sequence. A lambda gt 11 cDNA library derived from spinach leaves was screened using an antibody against NDP kinase I, which we previously purified to electrophoretic homogeneity (T. Nomura, T. Fukui, and A. Ichikawa, 1991, Biochim. Biophys. Acta 1077, 47-55). The cDNA sequences of positive clones contained the amino acid coding region (444 base pairs) for NDP kinase I as well as 5' and 3' noncoding regions of 33 and 361 base pairs, respectively. The cDNAs hybridized to a 1.1-kb mRNA. NDP kinase I contains 148 amino acid residues with a molecular mass of 16,305, which is in excellent agreement with that of the purified enzyme (16 kDa). Homology was found between the sequence of spinach NDP kinase I and those of the rat, Myxococcus xanthus, and Dictyostelium discoideum NDP kinases, as well as the human Nm23-gene product and the awd protein of Drosophila melanogaster.     

Interactions between Escherichia coli nucleoside-diphosphate kinase and DNA.

Nucleoside-diphosphate (NDP) kinase (NTP:nucleoside-diphosphate phosphotransferase) catalyzes the reversible transfer of gamma-phosphates from nucleoside triphosphates to nucleoside diphosphates through an invariant histidine residue. It has been reported that the high-energy phosphorylated enzyme intermediate exhibits a protein phosphotransferase activity toward the protein histidine kinases CheA and EnvZ, members of the two-component signal transduction systems in bacteria. Here we demonstrate that the apparent protein phosphotransferase activity of NDP kinase occurs only in the presence of ADP, which can mediate the phosphotransfer from the phospho-NDP kinase to the target enzymes in catalytic amounts (approximately 1 nm). These findings suggest that the protein kinase activity of NDP kinase is probably an artifact attributable to trace amounts of contaminating ADP. Additionally, we show that Escherichia coli NDP kinase, like its human homologue NM23-H2/PuF/NDP kinase B, can bind and cleave DNA. Previous in vivo functions of E. coli NDP kinase in the regulation of gene expression that have been attributed to a protein phosphotransferase activity can be explained in the context of NDP kinase-DNA interactions. The conservation of the DNA binding and DNA cleavage activities between human and bacterial NDP kinases argues strongly for the hypothesis that these activities play an essential role in NDP kinase function in vivo.     

Nucleoside diphosphate kinases in mammalian signal transduction systems: recent development and perspective

The role of nucleoside diphosphate (NDP) kinase with special reference to mammalian signal transduction systems was described. The interaction between NDP kinases and G proteins was reevaluated in view of their protein structural information and its significance was extended further on the basis of recent findings obtained with small molecular weight G proteins such as Rad, menin, and Rac. Meanwhile, observations suggesting involvement of NDP kinases in the regulation of cell growth and differentiation led to the realization that NDP kinases may play a crucial role in receptor tyrosine kinase signal transduction systems. In fact, a number of experimental results, particularly obtained with PC12 cells, implicate that NDP kinases appear to regulate differentiation marker proteins and cell-cycle-associated proteins cooperatively. Consequently, we propose a hypothesis that NDP kinases might act like a molecular switch to determine the cell fate toward proliferation or differentiation in response to environmental signals.     

Nm23-H1/NDP kinase folding intermediates and cancer: a hypothesis

The Nm23-H1/nucleoside diphosphate (NDP) kinase A is a metastasis suppressor, besides its enzymatic activity. The mutant S120G has been found in high-grade neuroblastomas. The mutant protein, once denatured in urea, is unable to refold in vitro. A size-exclusion chromatography analysis of the folding/association pathway showed that recombinant wild-type and S120G mutant human Nm23-H1/NDP kinase A unfold and refold passing through a molten globule state while typical hexameric NDP kinases unfold without dissociated species and refold through a native monomeric intermediate. A survey of the recent literature showed that several proteins involved in cancer, and their mutants, are marginally stable, like the wild-type Nm23-H1/NDP kinase A, or are misfolded, like its S120G mutant. We therefore suggest that the low thermodynamic stability and the folding intermediate of the Nm23-H1/NDP kinase A may be necessary for its regulatory properties.     

A Nucleoside Diphosphate Kinase from Paramecium tetraurelia with Protein Kinase Activity

Nucleoside diphosphate kinase (NDP kinase) from Paramecium was purified to homogeneity. The native enzyme was 80 kDa (by gel filtration), with subunits of 18 and 20 kDa. Near the amino terminus, 15 of 20 residues were identical with those in human NDP kinase, and 17 of 20 with the awd gene product from Drosophila. NDP kinase bound α-labeled ATP and GTP, and a photoreactive GTP analog labeled both subunits. Purified NDP kinase underwent autophosphorylation on a histidine and a serine residue using either ATP or GTP as a substrate. The enzyme also catalyzed acid-stable phosphorylation of casein and phosvitin. This protein kinase activity is distinct from the histidine phosphorylation that is part of the NDP kinase catalytic cycle. Antiserum against the purified protein from Paramecium cross-reacted with 16- to 20-kDa proteins in most species tested, and with a larger protein (44 kDa) in Paramecium, Xenopus, and two human lines. The multiple forms (20 and 44 kDa) of the NDP kinase in Paramecium and its protein kinase activity, suggest that the protein is more than a housekeeping enzyme; it may have regulatory roles such as those of the NDP kinase-like awd protein of Drosophila and Nm23 protein of humans.     

Structural and functional features of an NDP kinase from the hyperthermophile crenarchaeon Pyrobaculum aerophilum

Nucleoside diphosphate (NDP) kinases are ubiquitous enzymes that transfer gamma-phosphates from nucleoside triphosphates to nucleoside diphosphates via a ping-pong mechanism. The important role of this large family of enzymes in controlling cellular functions and developmental processes along with their crystallizability has made them good candidates for structural studies. We recently determined the structure of an evolved version of an NDP kinase from Pyrobaculum aerophilum, an extreme thermophile. This NDP kinase has similarity to the 42 other NDP kinases deposited in the Protein Data Bank (PDB) but differs significantly in sequence, structure, and biophysical properties. The P. aerophilum NDP kinase sequence contains two unique segments not present in other NDP kinases, comprising residues 66-100 and 156-165. We show that deletion mutants of the P. aerophilum NDP kinase lacking either or both of these inserts have an altered substrate specificity, allowing dGTP as the phosphate donor. A structural analysis of the evolved NDP kinase in conjunction with mutagenesis experiments suggests that the substrate specificity of the P. aerophilum NDP kinase is related to the presence of these two inserts.     

Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: purification to high specific activity by a two-step affinity chromatography procedure

This report describes a two-column scheme for purifying a pyrimidine nucleoside monophosphate kinase from rat bone marrow cells. Purification was achieved by affinity chromatography on Blue Sepharose and cellulose phosphate, with selective elution of the enzyme by substrates (UMP, ATP). The enzyme preparation appeared to be about 90% pure upon polyacrylamide gel electrophoresis, exhibited an exceptionally high specific activity (greater than 600 mumol/min/mg protein), and was obtained with 30-36% recovery of enzyme activity. It was concluded that UMP, dUMP, and CMP serve as phosphate acceptors for the enzyme, based on the parallel behavior displayed by enzyme activity with these substrates both during the purification process and during other procedures. The purified enzyme preparation did not display dTMP kinase activity. This report also describes a simplified radiotracer assay for pyrimidine nucleoside monophosphate kinases. Thin-layer chromatography on polyethyleneimine-cellulose is used to resolve residual substrates and products. Because both nucleoside di- and triphosphates remain at the origin, the assay is insensitive to the action of nucleoside diphosphate kinases and does not require the use of marker compounds. A variety of radiolabeled substrates can be used with this assay, including UMP, dUMP, CMP, and dTMP.     

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.     

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.     

Characterization of a low molecular mass autophosphorylating protein in cultured sugarcane cells and its identification as a nucleoside diphosphate kinase.

A low molecular mass (18 kD) phosphoprotein (pp18) was characterized and purified from cultured sugarcane (Saccharum officinarum L.) cell line H50-7209. Autophosphorylation assays were used to detect pp18 after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Only pp18 was detected by a brief in situ phosphorylation method, whereas additional putative protein kinases were detected by an extended method. pp18 was present in both microsomal membrane and soluble fractions and exhibited anomalous turnover of 32P label during in vitro phosphorylation experiments with highest levels present at shorter incubation times. Two major isoforms of the protein were identified in two-dimensional isoelectric focusing/SDS-PAGE of crude extracts and microsomal fractions. The levels of pp18 were enhanced approximately 4-fold by heat shock at 36 degrees C and the elevated pp18 decayed after heat shock was discontinued. pp18 was purified to apparent homogeneity, could be phosphorylated on serine residues, and also exhibited kinase-like activity toward histone H1. The amino acid sequence obtained from a cyanogen bromide digest was greater than 80% identical to nucleoside diphosphate (NDP) kinases from a variety of organisms. Biochemical analysis of the purified protein confirmed the identity as NDP kinase. Thus, NDP kinase appears to be modulated by heat shock in plants.