Residue 134 determines the dimer-tetramer assembly of nucleoside diphosphate kinase from moderately halophilic bacteria

Halomonas nucleoside diphosphate kinase (HaNDK) forms a dimeric assembly and Pseudomonas NDK (PaNDK) forms a tetrameric assembly. The mutation of Glu134 to Ala in HaNDK resulted in the conversion of the native dimeric structure to the tetramer assembly. Conversely, the mutation of Ala134 to Glu in PaNDK lead to the conversion from the tetramer to the dimer assembly, indicating that a single amino acid substitution at position 134 results in an alteration of the oligomeric structure of NDK. By modeling the structure of HaNDK and PaNDK based on the crystal structure of Myxococcus NDK, we showed that Glu134 exerts sufficient repulsive forces to disrupt the dimer-dimer interaction and prevent the formation of the tetramer.     

Killing of target cells by redirected granzyme B in the absence of perforin

We have previously reported that nucleoside diphosphate kinase (HsNDK) from extremely halophilic archaeon Halobacterium salinarum was expressed in Escherichia coli as a soluble, but inactive form and required high salt concentrations for in vitro folding and activation. Here, we found that fusion of extra sequence containing hexa-His-tag at amino-terminus of HsNDK (His-HsNDK) facilitated folding and activation of HsNDK in E. coli. This is a first observation of active folding of halophilic enzyme from extremely halophilic archaeon in E. coli. The in vitro refolding rate of His-HsNDK after heat denaturation was greatly increased over the native HsNDK. Folded His-HsNDK isolated from E. coli formed a hexamer in both 0.2 M and 3.8 M NaCl at 30 °C, while the native HsNDK purified from H. salinarum dissociated to dimer in 0.2 M NaCl. The observed hexameric structure in 0.2 M NaCl indicates that amino-terminal extension also enhances dimer to hexamer assembly and stabilizes the structure in low salt. These results suggest that positive charges in fused amino-terminal extension are effective in suppressing the negative charge repulsion of halophilic enzyme and thus, facilitate folding and assembly of HsNDK.     

Secondary and quaternary structural transition of the halophilic archaeon nucleoside diphosphate kinase under high- and low-salt conditions

Most halophilic enzymes from extremely halophilic archaea are denatured immediately after transfer from high-salt to low-salt medium. However, nucleoside diphosphate kinase (HsNDK) from the extremely halophilic archaeon Halobacterium salinarum seems to be exceptional, since the enzyme exhibited catalytic activity even under the low-salt condition. Here we show the mechanism how HsNDK is active under both high- and low-salt conditions that the HsNDK hexamer in high-salt medium dissociates into a dimer in the low-salt medium without denaturation. The observed change of the subunit structure was accompanied by a large decrease of alpha-helical content and lowered thermal sensitivity, yet keeping the conformations. This novel hexamer to dimer conversion under high- and low-salt conditions, respectively, seems to be the mechanism by which HsNDK is avoided from the irreversible denaturation.     

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.     

Dimeric structure of nucleoside diphosphate kinase from moderately halophilic bacterium: contrast to the tetrameric Pseudomonas counterpart

Light scattering and chemical cross-linking analyses of nucleoside diphosphate kinase (NDK) from moderate halophile, Halomonas sp. 593 (HaNDK), unambiguously demonstrated that this enzyme formed a dimeric structure, in contrast to the Pseudomonas NDK (PaNDK), a nonhalophilic counterpart, and other NDKs from Gram-negative bacteria, which all formed a tetrameric structure. Comparison of HaNDK and PaNDK showed that the HaNDK was less thermally stable than the PaNDK: the optimum temperature of PaNDK enzyme activity was 20 degrees C higher than that of HaNDK. However, the HaNDK readily refolded and reassembled back to the active dimeric structure, upon heat denaturation at 0.2 M NaCl, as soon as the temperature was lowered. On the contrary, the thermally more stable PaNDK was irreversibly denatured at its melting temperature.     

Characterization of a cytosolic nucleoside diphosphate kinase associated with cell division and growth in potato

A cDNA encoding Solanum chacoense cytosolic NDPK (NDPK1, EC 2.7.4.6) was isolated. The open reading frame encoded a 148 amino acid protein that shares homology with other cytosolic NDPKs including a conserved N-terminal domain. S. chacoense NDPK1 was expressed in Escherichia coli as a 6×His-tagged protein and purified by affinity chromatography. The recombinant protein exhibited a pattern of abortive complex formation suggesting that the enzyme is strongly regulated by the NTP/NDP ratio. A polyclonal antibody generated against recombinant NDPK1 was specific for the cytosolic isoform in Solanum tuberosum as shown from immunoprecipitation experiments and immunoblot analysis of chloroplasts and mitochondria preparations. NDPK activity and NDPK1 protein were found at different levels in various vegetative and reproductive tissues. DEAE fractogel analyses of NDPK activity in root tips, leaves, tubers and cell cultures suggest that NDPK1 constitutes the bulk of extractable NDPK activity in all these organs. NDPK activity and NDPK1 protein levels raised during the exponential growth phase of potato cell cultures whereas no rise in activity or NDPK1 protein was observed when sucrose concentration in the culture was manipulated to limit growth. Activity measurements, immunoblot analysis as well as immunolocalization experiments performed on potato root tips and shoot apical buds demonstrated that NDPK1 was predominantly localized in the meristematic zones and provascular tissues of the apical regions. These data suggest that NDPK1 plays a specific role in the supply of UTP during early growth of plant meristematic and provascular tissues.     

A single amino acid substitution changes the substrate specificity of quinoprotein glucose dehydrogenase in Gluconobacter oxydans.

Summary Gluconobacter oxydans contains pyrroloquinoline quinone-dependent glucose dehydrogenase (GDH). Two isogenic G. oxydans strains, P1 and P2, which differ in their substrate specificity with respect to oxidation of sugars have been analysed. P1 can oxidize only d-glucose, whereas P2 is also capable of the oxidation of the disaccharide maltose. To investigate the nature of this maltose-oxidizing property we cloned the gene encoding GDH from P2. Expression of P2 gdh in P1 enables the latter strain to oxidize maltose, indicating that a mutation in the P2 gdh gene is responsible for the change in substrate specificity. This mutation could be ascribed to a 1 by substitution resulting in the replacement of His 787 by Asn.     

Mitochondrial nucleoside diphosphate kinase: Mode of interaction with the outer mitochondrial membrane and proportion of catalytic activity functionally coupled to oxidative phosphorylation

In the present study, we found that ionic interactions are not essential for the binding of nucleoside diphosphate kinase of liver mitochondria outer compartment to outer mitochondrial membrane and that the proportion of the enzyme activity involved in functional coupling with oxidative phosphorylation (we demonstrated the existence of functional coupling earlier) is only 17%. Additional evidence was obtained that functionally coupled activity of nucleoside diphosphate kinase is associated with the outer surface of mitochondria. Dextran (10%) did not increase functional coupling. The physological importance of these effects is discussed. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 3, pp. 395–407.     

Localization and characterization of the mitochondrial isoform of the nucleoside diphosphate kinase in the pancreatic beta cell: evidence for its complexation with mitochondrial succinyl-CoA synthetase

Nucleoside diphosphate kinase (NDPK) catalyzes the transfer of terminal phosphates from nucleoside triphosphates to nucleoside diphosphates to yield nucleotide triphosphates. The present study was undertaken to localize and characterize the mitochondrial isoform of NDPK (mNDPK) in the pancreatic beta cell since it could contribute to the generation of mitochondrial nucleotide triphosphates and, thereby, to the mitochondrial high-energy phosphate metabolism of the pancreatic beta cell. Mitochondrial fractions from the insulin-secreting beta cells were isolated by differential centrifugation. mNDPK activity was assayed as the amount of [(3)H]GTPgammaS formed from ATPgammaS and [(3)H]GDP. Incubation of isolated mitochondrial extracts with either [gamma-(32)P]ATP or GTP resulted in the formation [(32)P]NDPK, which could be immunoprecipitated by an anti-NDPK serum. mNDPK exhibited saturation kinetics with respect to its nucleoside diphosphate acceptors and nucleoside triphosphate donors and sensitivity to known inhibitors of NDPK (e.g., uridine diphosphate and cromoglycate). By Western blot analyses, at least three isoforms of NDPK were identified in various subcellular fractions of the beta cell. The nm23-H1 (NDPK-A) was predominantly soluble whereas nm23-H2 (NDPK-B) was associated with the soluble as well as membranous fractions. The mitochondrial isoform of NDPK, nm23-H4, was uniformly distributed in the beta cell mitochondrial subfractions. A significant amount of NDPK (as determined by the catalytic activity and immunological methods) was recovered in the immunoprecipitates of mitochondrial fraction precipitated with an antiserum directed against succinyl-CoA synthetase (SCS), suggesting that NDPK might remain complexed with SCS. We provide the first evidence for the localization of a mitochondrial isoform of the NDPK in the islet beta cell and thus offer a potential mechanism for the generation of intramitochondrial GTP which, unlike ATP, is not transported into mitochondria via the classical nucleotide translocase. Further work will be required to determine the importance of the NDPK/SCS complex to normal beta cell function in the secretion of insulin.     

A requirement for MAP kinase in the assembly and maintenance of the mitotic spindle

Circumstantial evidence has suggested the possibility of microtubule-associated protein (MAP) kinase's involvement in spindle regulation. To test this directly, we asked whether MAP kinase was required for spindle assembly in Xenopus egg extracts. Either the inhibition or the depletion of endogenous p42 MAP kinase resulted in defective spindle structures resembling asters or half-spindles. Likewise, an increase in the length and polymerization of microtubules was measured in aster assays suggesting a role for MAP kinase in regulating microtubule dynamics. Consistent with this, treatment of extracts with either a specific MAP kinase kinase inhibitor or a MAP kinase phosphatase resulted in the rapid disassembly of bipolar spindles into large asters. Finally, we report that mitotic progression in the absence of MAP kinase signaling led to multiple spindle abnormalities in NIH 3T3 cells. We therefore propose that MAP kinase is a key regulator of the mitotic spindle.     

A new member of the short-chain dehydrogenases/reductases superfamily: Purification, characterization and substrate specificity of a recombinant carbonyl reductase from Pichia stipitis

A novel short-chain dehydrogenases/reductases superfamily (SDRs) reductase (PsCR) from Pichia stipitis that produced ethyl (S)-4-chloro-3-hydroxybutanoate with greater than 99% enantiomeric excess, was purified to homogeneity using fractional ammonium sulfate precipitation followed by DEAE-Sepharose chromatography. The enzyme purified from recombinant Escherichia coli had a molecular mass of about 35 kDa on SDS–PAGE and only required NADPH as an electron donor. The K_m value of PsCR for ethyl 4-chloro-3-oxobutanoate was 4.9 mg/mL and the corresponding V_max was 337 μmol/mg protein/min. The catalytic efficiency value was the highest ever reported for reductases from yeasts. Moreover, PsCR exhibited a medium-range substrate spectrum toward various keto and aldehyde compounds, i.e., ethyl-3-oxobutanoate with a chlorine substitution at the 2 or 4-position, or α,β-diketones. In addition, the activity of the enzyme was strongly inhibited by SDS and β-mercaptoethanol, but not by ethylene diamine tetra acetic acid.     

Quantification of uridine 5'-diphosphate (UDP)-glucose by high-performance liquid chromatography and its application to a nonradioactive assay for nucleoside diphosphate kinase using UDP-glucose pyrophosphorylase as a coupling enzyme

We describe a method for the detection and quantification of nucleoside diphosphate kinase (NDPK). NDPK catalyzes the transfer of the gamma-phosphate of cytidine 5'-triphosphate on uridine 5'-diphosphate (UDP) to produce uridine 5'-triphosphate (UTP). The method uses a nonradioactive coupled enzyme assay in which UTP produced by NDPK is utilized by UDP-glucose pyrophosphorylase. This latter enzyme synthesizes UDP-glucose and inorganic phosphate in the presence of glucose 1-phosphate. UDP-glucose is detected at 260 nm after separation of the reaction mixture by high-performance liquid chromatography (HPLC) on a strong anion-exchange column. The assay is reliable, specific, and linear with respect to time and enzyme amount. Using 15 min incubation time, the method allows detection of NDPK activity below 10 pmol/min. It can be used to analyze kinetic behavior and to quantify NDPK from a wide variety of animal, microbial, and plant sources. It also provides an alternative to radiometric assays and an improvement on pyruvate kinase-linked spectrophotometric assays, which can be hampered by pigments present in crude extracts. Furthermore, we show that the HPLC method developed here can be directly used to assay enzymes for which UDP-glucose is a product.     

Rescue of the neuroblastoma mutant of the human nucleoside diphosphate kinase A/nm23-H1 by the natural osmolyte trimethylamine-N-oxide

The point mutation S120G in human nucleoside diphosphate kinase A, identified in patients with neuroblastoma, causes a protein folding defect. The urea-unfolded protein cannot refold in vitro, and accumulates as a molten globule folding intermediate. We show here that the trimethylamine-N-oxide (TMAO) corrects the folding defect and stimulated subunit association. TMAO also substantially increased the stability to denaturation by urea of both wild-type and S120G mutant. A non-native folding intermediate accumulated in the presence of 4.5-7 M urea and of 2 M TMAO. It was inactive, monomeric, contained some secondary structure but no tertiary structure and displayed a remarkable stability to denaturation.     

In the early phase of programmed cell death in Tobacco Bright Yellow 2 cells the mitochondrial adenine nucleotide translocator, adenylate kinase and nucleoside diphosphate kinase are impaired in a reactive oxygen species-dependent manner

To investigate whether and how mitochondria can change in plant programmed cell death (PCD), we used the non-photosynthetic Tobacco Bright Yellow 2 (TBY-2) cells. These can be synchronized to high levels, stand out in terms of growth rate and homogeneity and undergo PCD as a result of heat shock. Using these cells we investigated the activity of certain mitochondrial proteins that have a role in providing ATP and/or other nucleoside triphosphates (NTPs). We show that, already after 2 h from the heat shock, when cell viability remains unaffected, the rate of ADP/ATP exchange due to adenine nucleotide translocator (ANT) activity, and the rate of the reactions catalysed by adenylate kinase (ADK; EC 2.7.4.3) and nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) are inhibited in a non-competitive-like manner. In all cases, externally added ascorbate partially prevented the inhibition. These effects occurred in spite of minor (for ANT) or no changes in the mitochondrial protein levels as immunologically investigated. Interestingly, a decrease of both the steady state level of the ascorbate pool and of the activity of l-galactono-γ-lactone dehydrogenase (GLDH) (EC 1.3.2.3), the mitochondrial enzyme catalysing the last step of ascorbate biosynthesis, were also found.