Purification of a serine and histidine phosphorylated mitochondrial nucleoside diphosphate kinase from Pisum sativum.

For the first time, to our knowledge, a nucleoside diphosphate kinase (NDPK) has been purified from plant mitochondria (Pisum sativum L.). In intact pea leaf mitochondria, a 17.4-kDa soluble protein was phosphorylated in the presence of EDTA when [gamma-32P]ATP was used as the phosphate donor. Cell fractionation demonstrated that the 17.4-kDa protein is a true mitochondrial protein, and the lack of accessibility to EDTA of the matrix compartment in intact mitochondria suggested it may have an intermembrane space localization. The 17.4-kDa protein was purified from mitochondrial soluble proteins using ATP-agarose and anion exchange chromatography. Amino-acid sequencing of two peptides, resulting from a trypsin digestion, revealed high similarity with the conserved catalytic phosphohistidine site and with the C-terminal of NDPKs. Acid and alkali treatments of [32P]-labelled pea mitochondrial NDPK indicated the presence of acid-stable as well as alkali-stable phosphogroups. Thin-layer chromatography experiments revealed serine as the acid-stable phosphogroup. The alkali-stable labelling probably reflects phosphorylation of the conserved catalytic histidine residue. In phosphorylation experiments, the purified pea mitochondrial NDPK was labelled more heavily on serine than histidine residues. Furthermore, kinetic studies showed a faster phosphorylation rate for serine compared to histidine. Both ATP and GTP could be used as phosphate donor for histidine as well as serine labelling of the pea mitochondrial NDPK.     

Identification of a mitochondrial nucleoside diphosphate kinase from the green alga Dunaliella tertiolecta

We isolated a full-length cDNA encoding a nucleoside diphosphate (NDP) kinase from a Dunaliella tertiolecta cDNA library by homology cloning and rapid amplification of cDNA ends-PCR. The cDNA sequence, consisting of 840 bp, contained an open reading frame coding for a 221-amino acid protein. The predicted 24-kDa protein was named DtNDK1. It possesses all the residues involved in nucleotide binding and catalysis and, in its long N-terminus, contains putative mitochondrial targeting peptides. The full-length pre-protein expressed in Escherichia coli as a recombinant N-terminally His-tagged protein was retained in inclusion bodies, totally devoid of NDP kinase activity. Upon expression in yeast cells, the full-length protein His-tagged at the C-terminus was found processed in a soluble form that was lacking the first 67 amino acids from the N-terminus. The mature protein, which was purified by affinity chromatography to near homogeneity, showed NDP kinase activity. Confocal microscopy on yeast cells expressing the recombinant protein revealed the specific mitochondrial localization of DtNDK1 labeled at the C-terminus with green fluorescent protein.     

Identification and mutational analysis of amino acid residues involved in dipyridamole interactions with human and Caenorhabditis elegans equilibrative nucleoside transporters

The equilibrative nucleoside transporters, hENT1 and CeENT1 from humans and Caenorhabditis elegans, respectively, are inhibited by nanomolar concentrations of dipyridamole and share a common 11-transmembrane helix (TM) topology. Random mutagenesis and screening by functional complementation in yeast for clones with reduced sensitivities to dipyridamole yielded mutations at Ile429 in TM 11 of CeENT1 and Met33 in TM 1 of hENT1. Mutational analysis of the corresponding residues of both proteins suggested important roles for these residues in competitive inhibition of hENT1 and CeENT1 by dipyridamole. To verify the roles of these residues in dipyridamole interactions, hENT2, which naturally exhibits low dipyridamole sensitivity, was mutated to contain side chains favorable for high affinity dipyridamole binding (i.e. a Met at the TM 1 and/or an Ile at the TM 11 positions). The single mutants exhibited increased hENT2 sensitivity to inhibition by dipyridamole, and the double mutant was the most sensitive, with an IC50 value that was only 2% of that of wild type. Functional analysis of the TM 1 and 11 mutants of hENT1 and CeENT1 revealed that Ala and Thr in the TM 1 and 11 positions, respectively, impaired uridine and adenosine transport and that Leu442 of hENT1 was involved in permeant selectivity. Mechanistic and structural models of dipyridamole interactions with the TM 1 and 11 residues are proposed. This study demonstrated that the corresponding residues in TMs 1 and 11 of hENT1, hENT2, and CeENT1 are important for dipyridamole interactions and nucleoside transport.     

Mutational analysis of nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of critical amino acid residues involved in exopolysaccharide alginate synthesis.

We report the utilization of site-directed and random mutagenesis procedures in the gene encoding nucleoside diphosphate kinase (ndk) from Pseudomonas aeruginosa in order to examine the role of Ndk in the production of alginate by this organism. Cellular levels of the 16-kDa form of the Ndk enzyme are greatly reduced in P. aeruginosa 8830 with a knockout mutation in the algR2 gene (8830R2::Cm); this strain is also defective in the production of the exopolysaccharide alginate. In this study, we isolated four mutations in ndk (Ala-14-->Pro [Ala14Pro], Gly21Val, His117Gln, and Ala125Arg) which resulted in the loss of Ndk biochemical activity; hyperexpression of any of these four mutant genes did not restore alginate production to 8830R2::Cm. We identified six additional amino acid residues (Ser-43, Ala-56, Ser-69, Glu-80, Gly-91, and Asp-135) whose alteration resulted in the inability of Ndk to complement alginate production. After hyperproduction in 8830R2::Cm, it was determined that each of these six mutant Ndks was biochemically active. However, in four cases, the in vivo levels of Ndk were reduced, which consequently affected the growth of 8830R2::Cm in the presence of Tween 20. Two mutant Ndk proteins which could not complement the alginate synthesis defect in 8830R2::Cm were not affected in any characteristic examined in the present study. All of the mutant Ndks characterized which were still biochemically active formed membrane complexes with Pk, resulting in GTP synthesis. Two of the four Ndk activity mutants (His117Gln and Ala125Arg) identified were capable of being truncated to 12 kDa and formed a membrane complex with Pk; however, the complexes formed were inactive for GTP synthesis. The other two Ndk activity mutants could be truncated to 12 kDa but were not detected in membrane fractions. These results further our understanding of the role of Ndk in alginate synthesis and identify amino acid residues in Ndk which have not previously been studied as critical to this process.     

Structure of a halophilic nucleoside diphosphate kinase from Halobacterium salinarum

Nucleoside diphosphate kinase from the halophilic archaeon Halobacterium salinarum was crystallized in a free state and a substrate-bound form with CDP. The structures were solved to a resolution of 2.35 and 2.2A, respectively. Crystals with the apo-form were obtained with His6-tagged enzyme, whereas the untagged form was used for co-crystallization with the nucleotide. Crosslinking under different salt and pH conditions revealed a stronger oligomerization tendency for the tagged protein at low and high salt concentrations. The influence of the His6-tag on the halophilic nature of the enzyme is discussed on the basis of the observed structural properties.     

The pea mitochondrial nucleoside diphosphate kinase cleaves DNA and RNA

Here, we present the characterization of a plant NDPK exhibiting nuclease activity. This is the first identification of a nuclease localised in the intermembrane space of plant mitochondria. The recombinant pea NDPK3 protein cleaves not only supercoiled plasmid DNA, but also highly structured RNA molecules such as tRNAs or the 3'UTR of the atp9 mRNA suggesting that the NDPK3 nuclease activity has a structural requirement. ATP inhibits this nuclease activity, while ADP has no effect. Furthermore, studies on NDPK mutant proteins indicate that the nuclease- and the kinase-mechanisms are separate.     

Amino acid residues involved in autophosphorylation and phosphotransfer activities are distinct in nucleoside diphosphate kinase from Mycobacterium tuberculosis

Nucleoside diphosphate kinase (NdK) is a ubiquitous enzyme in both prokaryotes and eukaryotes and is primarily involved in the maintenance of cellular nucleotide pools. We have cloned ndk from Mycobacterium tuberculosis strain H37Ra and expressed it in Escherichia coli as a fusion protein with glutathione S-transferase. The purified protein, following thrombin cleavage and gel permeation chromatography, was found to be hexameric with a monomeric unit molecular mass of approximately 16.5 kDa. The protein exhibited nucleotide binding, divalent cation-dependent autophosphorylation, and phosphate transfer ability from nucleoside triphosphate to nucleoside diphosphate. Although UDP inhibited the catalytic activity of the recombinant protein, the classic inhibitors, like cromoglycate, 5'-adenosine 3'-phosphate, and adenosine 3'-phosphate 5'-phosphosulfate, had no effect on the activity. Among three histidine residues in the protein, His-117 was found to be essential for autophosphorylation. However, in subsequent phosphate transfer, we observed that His-53 had a significant contribution. Consistent with this observation, substitution of His-53 with either Ala or Gln affected the ability of the recombinant protein to complement NdK function in Pseudomonas aeruginosa. Furthermore, mutational analysis established critical roles for Tyr-50 and Arg-86 of the M. tuberculosis protein in maintaining phosphotransfer ability.     

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.     

Identification of serine phosphorylation in mitochondrial uncoupling protein 1

Native uncoupling protein 1 was purified from rat brown adipose tissue of cold-acclimated rats and rats kept at room temperature, in the presence of phosphatase inhibitors. The purified protein from cold-acclimated animals was digested with trypsin and immobilized metal affinity chromatography was used to select for phosphopeptides. Tandem mass spectroscopic analysis of the peptides derived from uncoupling protein 1, suggests phosphorylation of serine 3 or 4 and identified phosphorylation of serine 51. Furthermore, we were able to demonstrate that antibodies to phosphoserine detect full-length UCP 1 and that the proportion of phosphoserine on UCP1, purified from cold-acclimated rats, was significantly greater than that on UCP 1 from rats kept at room temperature (90+/-4% compared to 62+/-8%, p=0.013), respectively). We conclude that uncoupling protein 1 is a phosphoprotein and that cold-acclimation increases the proportion of UCP1 that is serine phosphorylated.     

A fluorescent sensor of the phosphorylation state of nucleoside diphosphate kinase and its use to monitor nucleoside diphosphate concentrations in real time

A sensor for purine nucleoside diphosphates in solution based on nucleoside diphosphate kinase (NDPK) has been developed. A single cysteine was introduced into the protein and labeled with the environmentally sensitive fluorophore, N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide. The resultant molecule shows a 4-fold fluorescence increase when phosphorylated on His117; this phosphorylation is on the normal reaction pathway of the enzyme. The emission maximum of the phosphoenzyme is at 475 nm, with maximum excitation at 430 nm. The fluorescent phosphorylated NDPK is used to measure the amount of ADP and the unphosphorylated to measure ATP. The labeled protein is phosphorylated to > 90%, and the resultant molecule is stable on ice or can be stored at -80 degrees C. The fluorescence responds to the fraction of protein phosphorylated and so to the equilibrium between ADP plus NDPK approximately P and ATP plus NDPK. In effect, the sensor measures the ADP/ATP concentration ratio. The enzyme has a broad specificity for the purine of the nucleotides, so the sensor also can measure GDP/GTP ratios. The fluorescence and kinetic properties of the labeled protein are described. The binding rate constants of nucleotides are approximately 10(5) M(-1) s(-1), and the fluorescence change is at >200 s(-1) when the ADP concentration is >1 mM. Results are presented with two well-defined systems, namely, the kinetics of ADP release from myosin subfragment 1 and GDP release from the small G protein, human rho. The results obtained with this novel sensor agree with those from alternate methods and demonstrate the applicability for following micromolar changes in nucleoside diphosphate in real time.     

Identification of the residues involved in the unique serine specificity of Caenorhabditis elegans mitochondrial EF-Tu2

In canonical translation systems, the single elongation factor Tu (EF-Tu) recognizes all elongator tRNAs. However, in Caenorhabditis elegans mitochondria, two distinct EF-Tu species, EF-Tu1 and EF-Tu2, recognize 20 species of T armless tRNA and two species of D armless tRNA(Ser), respectively. We previously reported that C. elegans mitochondrial EF-Tu2 specifically recognizes the serine moiety of serylated-tRNA. In this study, to identify the critical residues for the serine specificity in EF-Tu2, several residues in the amino acid binding pocket of bacterial EF-Tu were systematically replaced with corresponding EF-Tu2 residues, and the mutants were analyzed for their specificity for esterified amino acids attached to tRNAs. In this way, we obtained a bacterial EF-Tu mutant that acquired serine specificity after the introduction of 10 EF-Tu2 residues into its amino acid binding pocket. C. elegans EF-Tu2 mutants lacking serine specificity were also created by replacing seven or eight residues with bacterial residues. Further stressing the importance of these residues, we found that they are almost conserved in EF-Tu2 sequences of closely related nematodes. Thus, these three approaches reveal the critical residues essential for the unique serine specificity of C. elegans mitochondrial EF-Tu2.     

The human nm23-H4 gene product is a mitochondrial nucleoside diphosphate kinase

We demonstrate here the catalytic activity and subcellular localization of the Nm23-H4 protein, product of nm23-H4, a new member of the human nm23/nucleoside diphosphate (NDP) kinase gene family (Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and Lacombe, M. L. (1997) Hum. Genet. 99, 550-557). Nm3-H4 was synthesized in escherichia coli as the full-length protein and as a truncated form missing the N-terminal extension characteristic of mitochondrial targeting. The truncated form possesses NDP kinase activity, whereas the full-length protein is inactive, suggesting that the extension prevents enzyme folding and/or activity. X-ray crystallographic analysis was performed on active truncated Nm23-H4. Like other eukaryotic NDP kinases, it is a hexamer. Nm23-H4 naturally possesses a serine residue at position 129, equivalent to the K-pn mutation of the Drosophila NDP kinase. The x-ray structure shows that the presence of Ser(129) has local structural effects that weaken subunit interactions. Site-directed mutagenesis shows that the serine is responsible for the lability of Nm23-H4 to heat and urea treatment, because the S129P mutant is greatly stabilized. Examination of human embryonic kidney 293 cells transfected with green fluorescent protein fusions by confocal microscopy shows a specific mitochondrial localization of Nm23-H4 that was also demonstrated by Western blot analysis of subcellular fractions of these cells. Import into mitochondria is accompanied by cleavage of the N-terminal extension that results in NDP kinase activity. Submitochondrial fractionation indicates that Nm23-H4 is associated with mitochondrial membranes, possibly to the contact sites between the outer and inner membranes.     

The phosphorylation status of membrane-bound nucleoside diphosphate kinase in epithelia and the role of AMP

Nucleoside diphosphate kinase (NDPK) has many roles and is present in different locations in the cell. Membrane-bound NDPK is present in epithelial fractions enriched for the apical membrane. Here, we show in human, mouse and sheep airway membranes, that the phosphorylation state of membrane-bound NDPK on histidine and serine residues differs dependent on many regulatory factors. GTP (but not ATP) promotes serine phosphorylation (pSer) of NDPK. Further we find that rising [AMP] promotes pSer (only with GTP) but inhibits histidine phosphorylation (pHis) of NDPK from both donors. We find that NDPK co-immunoprecipitates reciprocally with AMP-activated kinase and that these two proteins can co-localise in human airways. AMP concentrations rise rapidly when ATP is depleted or during hypoxia. We find that, in human airway cells exposed to hypoxia (3% oxygen), membrane-bound NDPK is inhibited. Although histidine phosphorylation should in principle be independent of the nucleotide triphosphates used, we speculate that this membrane pool of NDPK may be able to switch function dependent on nucleotide species.     

Heat shock modulates phosphorylation status and activity of nucleoside diphosphate kinase in cultured sugarcane cells

Nucleoside diphosphate kinase (NDPK) is involved in the regeneration of nucleoside triphosphates (NTPs) through its phosphotransferase activity via an autophosphorylating histidine residue. Additionally, autophosphorylation of serine and/or threonine residues is documented for NDPKs from various organisms. However, the metabolic significance of serine/threonine phosphorylation has not been well characterized. In this study we report the cloning and characterization of NDPKI from cultured sugarcane (Saccharum officinarum L. line H50-7209) cells, and modulation of serine autophosphorylation of NDPK1 in response to heat-shock (HS). Heat-shock treatment at 40°C for 2 h resulted in a 40% reduction in labeled phosphoserine in NDPK1. This dephosphorylation was accompanied by an increase in NDPK enzyme activity. In contrast, NDPK1 in cultured tobacco (cv. W-38) cells did not show changes in autophosphorylation or increased enzyme activity in response to HS. The mRNA or protein level of NDPK1 did not increase in response to HS. Sugarcane cells sustain the constitutive protein synthesis in addition to heat-shock protein synthesis during HS, while constitutive protein synthesis is significantly reduced in tobacco cells during HS. Thus, HS modulation of NDPK1 activity and serine dephosphorylation in sugarcane cells may represent an important physiological role in maintaining cellular metabolic functions during heat stress.     

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.     

Regulation of plant symbiosis receptor kinase through serine and threonine phosphorylation

We studied the biochemical properties of a plant receptor-like kinase to gain insights into the regulatory mechanism of this largest class of plant kinases. SYMRK (symbiosis receptor kinase) is required for early signal transduction leading to plant root symbioses with nitrogen-fixing rhizobia and phosphate-acquiring arbuscular mycorrhizal fungi. Amino acid substitutions in positions critical for activity of other related kinases cause a nonsymbiotic plant phenotype, suggesting that SYMRK kinase activity is required for symbiosis. SYMRK is capable of intermolecular autophosphorylation. Nonphosphorylated SYMRK is less active than the phosphorylated version, suggesting the phosphorylation status of SYMRK determines its activity. Three Ser/Thr residues were identified as residues required for full kinase activation through targeted mutagenesis. Using quadrupole time-of-flight mass spectrometry analysis, two of these were confirmed to be phosphorylated in vitro. These crucial phosphorylation sites are conserved among various plant receptor-like kinases as well as animal Pelle/interleukin-1 receptor associated kinase. Despite the distinct domain architecture of receptor-like kinases versus Pelle/interleukin-1 receptor associated kinase, our results suggest the existence of conserved activation mechanisms.