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.     

Biosynthesis of nucleoside analogues via thermostable nucleoside phosphorylase

Biocatalyzed synthesis of nucleoside analogues was carried out using two thermostable nucleoside phosphorylases from the hyperthermophilic aerobic crenarchaeon Aeropyrum pernix K1. The synthesis of the 2,6-diaminopurine nucleoside and 5-methyluridine was used as a reaction model to test the process. Both the purine nucleoside phosphorylase (apPNP) and uridine phosphorylase (apUP) were functionally expressed in Escherichia coli. The recombinant enzymes were characterized after purification, and both enzymes showed high thermostability and broad substrate specificity. Both enzymes retained 100 % of their activity after 60 min at high temperature, and the optimum temperature for the enzymes was 90–100 °C. The nucleoside phosphorylases obtained from A. pernix are valuable industrial biocatalysts for high-temperature reactions that produce nucleoside drugs in high yields.     

Studies on disaccharide nucleoside synthesis. Mechanism of the formation of trisaccharide purine nucleosides.

The unexpected formation of trisaccharide nucleosides during synthesis of purine 5'-O-beta-D-ribofuranosylnucleosides in the presence of Lewis acids was observed.     

Studies of nucleoside transporters using novel autofluorescent nucleoside probes

To better understand nucleoside transport processes and intracellular fates of nucleosides, we have developed a pair of fluorescent nucleoside analogues, FuPmR and dFuPmR, that differ only in the sugar moiety (ribofuranosyl versus 2'-deoxy, respectively), for real-time analysis of nucleoside transport into living cells by confocal microscopy. The binding and transportability of the two compounds were assessed for five recombinant human nucleoside transporters (hENT1/2, hCNT1/2/3) produced in Saccharomyces cerevisiae and/or oocytes of Xenopus laevis. The ribosyl derivative (FuPmR) was used to demonstrate proof of principle in live cell imaging studies in 11 cultured human cancer cell lines with different hENT1 activities. The autofluorescence emitted from FuPmR enabled direct visualization of its movement from the extracellular medium into the intracellular compartment of live cells, and this process was blocked by inhibitors of hENT1 (nitrobenzylmercaptopurine ribonucleoside, dipyridamole, and dilazep). Quantitative analysis of fluorescence signals revealed two stages of FuPmR uptake: a fast first stage that represented the initial uptake rate (i.e., transport rate) followed by a slow long-lasting second stage. The accumulation of FuPmR and/or its metabolites in nuclei and mitochondria was also visualized by live cell imaging. Measurements of fluorescence intensity increases in nuclei and mitochondria revealed rate-limited processes of permeant translocation across intracellular membranes, demonstrating for the first time the intracellular distribution of nucleosides and/or nucleoside metabolites in living cells. The use of autofluorescent nucleosides in time-lapse confocal microscopy is a novel strategy to quantitatively study membrane transport of nucleosides and their metabolites that will provide new knowledge of nucleoside biology.     

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.     

Inhibition of nucleoside Q formation in transfer ribonucleic acid during methionine starvation of relaxed-control Escherichia coli.

The elution profiles of Asp-tRNA from unstarved and starved cultures of a relaxed-control (Rel-) strain of Escherichia coli were compared by reversed-phase chromatography. Methionine starvation results in the appearance of several additional species of Asp-tRNA which are not observed with starvation for leucine or histidine. By the criterion of cyanogen bromide-effected shifts in chromatographic elution position, a large portion of the tRNAAsp synthesized in methionine-starved cells lacks the normal Q nucleoside. By the same criterion, virtually all of the tRNAAsp from unstarved, leucine-starved, and histidine-starved cells contain Q. We conclude that methionine starvation prevents the formation of the norma Q nucleoside in Rel- E. coli.     

Recent studies on nucleoside H-phosphonothioate and nucleoside methylphosphonothioate synthesis

The reaction of suitably protected nucleosides with phosphinic acid in the presence of condensing agents has been investigated and applied for the preparation of nucleoside 3'-H-phosphonothioates. Studies on synthesis and separation of diastereoisomers of nucleoside 3'-methylphosphonothioates are also discussed.     

Thermus thermophilus nucleoside phosphorylases active in the synthesis of nucleoside analogues

Cells extracts from Thermus thermophilus HB27 express phosphorolytic activities on purines and pyrimidine nucleosides. Five putative encoding genes were cloned and expressed in Escherichia coli, and the corresponding recombinant proteins were purified and studied. Two of these showed phosphorolytic activities against purine nucleosides, and third one showed phosphorolytic activity against pyrimidine nucleosides in vitro, and the three were named TtPNPI, TtPNPII, and TtPyNP, respectively. The optimal temperature for the activity of the three enzymes was beyond the water boiling point and could not be measured accurately, whereas all of them exhibited a wide plateau of optimal pHs that ranged from 5.0 to 7.0. Analytical ultracentrifugation experiments revealed that TtPNPI was a homohexamer, TtPNPII was a monomer, and TtPyNP was a homodimer. Kinetic constants were determined for the phosphorolysis of the natural substrates of each enzyme. Reaction tests with nucleoside analogues revealed critical positions in the nucleoside for its recognition. Activities with synthetic nucleobase analogues, such as 5-iodouracil or 2,6-diaminopurine, and arabinosides were detected, supporting that these enzymes could be applied for the synthesis of new nucleoside analogs with pharmacological activities.     

Sequence specificity of the non-natural pyrido[2,3-d]pyrimidine nucleoside in triple helix formation.

The non-natural pyrido[2,3-d]pyrimidine nucleoside F, which pairs preferentially with guanine (G) and adenine (A) within double-helical DNA, recognizes with high selectivity AT base pairs within triple-helical complexes. These observations suggest that F may exist in different tautomeric forms within double-helical and triple-helical complexes. Analysis of the base stacking properties of this extended ring system using two oligodeoxyribonucleotides containing terminal thymines and/or pyrido[2,3-d]pyrimidines bound to adjacent sites showed a decrease in free energy of binding in a triple-helical complex in the order (5'-3') TT > FT > TF > FF.     

Evidence for complex formation between GTP binding protein(Gs) and membrane-associated nucleoside diphosphate kinase

When the Gs in rat liver membranes was prelabeled with [32P]NAD and cholera toxin, solubilized with octylglucoside, and then analyzed by sucrose density gradient centrifugation, it was fractionated into two peaks with approximate molecular sizes of 12-13S and 3-4S. Pretreatment without or with GDP beta S of the labeled membranes resulted in a larger peak in the high molecular weight region, whereas pretreatment with glucagon plus GTP gamma S caused almost equal peaks in both regions. The affinity-purified anti-nucleoside diphosphate (NDP) kinase antibodies only precipitated the Gs in high molecular weight region. Under the same condition, small but significant NDP kinase activity was associated with the high molecular weight Gs region although a large portion of the enzyme activity was recovered in fractions where it alone should appear (6.2S). Both Lubrol-PX and digitonin solubilized the Gs in forms insensitive to immunoprecipitation by anti-NDP kinase antibodies although the latter detergent was able to solubilize the Gs in a high molecular weight form, that is, a ternary glucagon-receptor-G protein complex. These results demonstrate that Gs and membrane-associated NDP kinase may exist in part in a complexed form in membranes. Physiological relevance of the complex formation in membrane signal transduction is discussed.     

Purine nucleoside synthesis, an efficient method employing nucleoside phosphorylases

An improved method for the enzymatic synthesis of purine nucleosides is described. Pyrimidine nucleosides were used as pentosyl donors and two phosphorylases were used as catalysts. One of the enzymes, either uridine phosphorylase (Urd Pase) or thymidine phosphorylase (dThd Pase), catalyzed the phosphorolysis of the pentosyl donor. The other enzyme, purine nucleoside phosphorylase (PN Pase), catalyzed the synthesis of the product nucleoside by utilizing the pentose 1-phosphate ester generated from the phosphorolysis of the pyrimidine nucleoside. Urd Pase, dThd Pase, and PN Pase were separated from each other in extracts of Escherichia coli by titration with calcium phosphate gel. Each enzyme was further purified by ion-exchange chromatography. Factors that affect the stability of these catalysts were studied. The pH optima for the stability of Urd Pase, dThd Pase, and PN Pase were 7.6, 6.5, and 7.4, respectively. The order of relative heat stability was Urd Pase greater than PN Pase greater than dThd Pase. The stability of each enzyme increased with increasing enzyme concentration. This dependence was strongest with dThd Pase and weakest with Urd Pase. Of the substrates tested, the most potent stabilizers of Urd Pase, dThd Pase, and PN Pase were uridine, 2'-deoxyribose 1-phosphate, and ribose 1-phosphate, respectively. Some general guidelines for optimization of yields are given. In a model reaction, optimal product formation was obtained at low phosphate concentrations. As examples of the efficiency of the method, the 2'-deoxyribonucleoside of 6-(dimethylamino)purine and the ribonucleoside of 2-amino-6-chloropurine were prepared in yields of 81 and 76%, respectively.     

An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside.

Acyclic nucleoside analogues with carboxamido- or nitro-substituted heterocyclic bases have been evaluated for their possible use as universal bases in oligodeoxynucleotides. The acyclic moiety endows the constructs with enough flexibility to allow good base stacking. The 5-nitroindazole analogue afforded the most stable duplexes among the acyclic derivatives with the least spread in Tm versus the four natural bases. In spite of the acyclic moiety, stabilities are comparable with those of duplexes incorporating the recently described 5-nitroindole nucleoside analogue, but considerably exceed those for the 3-nitropyrrole analogue.     

Synthesis of nucleoside phosphosulfates

We describe an efficient and scalable procedure for the chemical synthesis of nucleoside 5'-phosphosulfates (NPS) from nucleoside 5'-phosphorimidazolides and sulfate bis(tributylammonium) salt. Using this method we obtained various NPS with yields ranging from 70-90%, including adenosine 5'-phosphosulfate (APS) and 2',3'-cyclic precursor of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), which are the key intermediates in the assimilation and metabolism of sulfur in all living organisms.     

Brain nucleoside recycling

The rate limiting reactions of nucleotide synthesis are modulated by intracellular fluctuations of nucleoside triphosphate concentrations. This topic has been mostly studied at the level of the de novo nucleotide synthesis from simple precursors. However, there are districts, such as brain, which rely more heavily on the salvage of preformed purine and pyrimidine rings, mainly in the form of nucleosides. This raises the following question: how do these districts maintain the right balance between the purine and pyrimidine pools? We believe that it is now safe to state that a cross talk exists between the extra- and intracellular metabolism of purine and pyrimidine nucleosides in the brain. The extracellular space is the major site of nucleoside generation through successive dephosphorylations of released triphosphates, whereas brain cytosol is the major site of multiple phosphorylations of uptaken nucleosides at their 5′-position. Modulation of both extracellular nucleoside generation by membrane bound ectonucleotidases, and intracellular nucleoside phosphorylation by cytosolic kinases might contribute to maintain the right extra- and intracellular purine and pyrimidine nucleotide balance in the brain.