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PubMed1992

Synthesis of 3'-C-methyladenosine and 3'-C-methyluridine diphosphates and their interaction with the ribonucleoside diphosphate reductase from Corynebacterium nephridii.

Two nucleoside diphosphate analogs, 3'-C-methyl-ADP and 3'-C-methyl-UDP, have been tested as substrate and/or allosteric effectors using the adenosylcobalamin-dependent ribonucleoside diphosphate reductase of Corynebacterium nephridii. Neither analog was a substrate for the reductase. However, they did function as allosteric effectors and as inhibitors of the reduction of ADP and UDP, respectively. The nucleotide analogs did not stimulate the hydrogen exchange reaction between [5'-3H2]adenosylcobalamin and the solvent, indicating that the cleavage of the 3'-carbon-hydrogen bond is a prerequisite for the exchange reaction. A reinvestigation of the requirements for the exchange reaction revealed that the deoxyribonucleoside diphosphate products are very effective promoters of this reaction. Indeed, the deoxyribonucleoside diphosphates were found to be more effective in promoting the exchange reaction than the ribonucleoside diphosphate substrates. In contrast, the deoxyribonucleoside triphosphate effectors, dATP, dUTP, and dTTP, were only marginally effective as promoters of this reaction.

Adenosine Diphosphate↗
PubMed1978

Eukaryotic ribosomal proteins stimulate Escherichia coli stringent factor to synthesize guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate, 3'-diphosphate (ppGpp).

When supplemented with Escherichia coli stringent factor, 80S ribosomes from various sources failed to support guanosine tetra- and pentaphosphate ((p)ppGpp) synthesis. In contrast, ribosomal proteins from 80S, 60S or 40S particles (mouse embryos, rabbit reticulocytes) crossreacted with the E. coli stringent factor. Significant stimulation of (p)ppGpp synthesis was achieved with a concentration as low as 5 micrograms of ribosomal proteins/ml. These observations may provide additional crtieria to detect homologies between eukaryotic and prokaryotic ribosomal proteins.

Bacterial Proteins↗
PubMed1998

Molecular cloning, expression, and purification of undecaprenyl diphosphate synthase. No sequence similarity between E- and Z-prenyl diphosphate synthases.

Cloning of the gene for undecaprenyl diphosphate synthase was successful, providing the first primary structure for any prenyltransferase that catalyzes Z-prenyl chain elongation. A genomic DNA library of Micrococcus luteus B-P 26 was constructed in Escherichia coli, and the recombinant clones were grown on nylon membranes. The membrane was incubated directly by floating it on a reaction mixture containing radiolabeled isopentenyl diphosphate, nonlabeled farnesyl diphosphate, and Mg2+. Only the clones harboring plasmids encoding prenyltransferases could take up the substrates to synthesize and accumulate radiolabeled products inside the cells in amounts large enough to be detectable by autoradiography. Four positive colonies were found among about 4,000 bacterial colonies of the genomic DNA library. Two of them carried the gene for undecaprenyl diphosphate synthase, which catalyzes the Z-prenyl chain elongation, and the others carried the (all-E)-hexaprenyl diphosphate synthase genes (hexs-a and hexs-b; Shimizu, N., Koyama, T., and Ogura, K. (1998) J. Bacteriol. 180, 1578-1581). The undecaprenyl diphosphate synthase, which had a predicted molecular mass of 28.9 kDa, was overproduced in E. coli cells by applying a soluble expression system, and it was purified to near homogeneity. The deduced primary structure of the Z-prenyl chain-elongating enzyme is totally different from those of E-prenyl chain-elongating enzymes, which have characteristic conserved regions, including aspartate-rich motifs.

Alkyl and Aryl Transferases↗
PubMed2002

Phosphorylation of pyrimidine L-deoxynucleoside analog diphosphates. Kinetics of phosphorylation and dephosphorylation of nucleoside analog diphosphates and triphosphates by 3-phosphoglycerate kinase.

Anticancer and antiviral D- and L-nucleoside analogs are phosphorylated stepwise in the cells to the pharmacologically active triphosphate metabolites. We recently reported that in the last step, L-deoxynucleoside analog diphosphates are phosphorylated by 3-phosphoglycerate kinase (PGK). To explain the preference of PGK for L- over D-deoxynucleoside analog diphosphates, the kinetics of their phosphorylation were compared with the dephosphorylation of the respective triphosphates using recombinant human PGK. The results attributed favorable phosphorylation of L-deoxynucleoside analog diphosphates by PGK to differences in k(cat), which were consequences of varied orientations of the sugar and diphosphates in the catalytic site of PGK. The amino acids involved in the catalytic reaction of PGK (including Glu(344), Lys(220), and Asn(337)) were therefore mutated. The impact of mutations on the phosphorylation of L- and D-deoxynucleoside analog diphosphates was different from those on dephosphorylation of the respective triphosphates. This suggested that the interactions of the nucleoside analogs with amino acids during the transition state are different in the phosphorylation and dephosphorylation reactions. Thus, reversible action of the enzyme may not involve the same configuration of the active site. Furthermore, the amino acid determinants of the action of PGK for L-deoxynucleotides were not the same as for the D-deoxynucleotides. This study also suggests the potential impact of nucleoside analog diphosphates and triphosphates on the multiple cellular functions of PGK, which may contribute to the action of the analogs.

Amino Acid Substitution↗
PubMed1982

Energy deprivation and guanosine 5'-diphosphate 3'-diphosphate synthesis in cyanobacteria.

A reduction in the incident light intensity has been used to elicit guanosine 5'-diphosphate 3'-diphosphate accumulation in cyanobacteria. Inhibitors of photophosphorylation, 2,4-dinitrophenol, and carbonyl cyanide-m-chlorophenyl hydrazone elicited accumulation in three species of cyanobacteria when they were grown on dinitrogen or nitrate, but not in cultures grown on ammonium or glutamine. Accumulation of guanosine 5'-diphosphate 3'-diphosphate also preceded a substantial reduction of the purine nucleoside triphosphate pools. This accumulation of guanosine 5'-diphosphate 3'-diphosphate is therefore not primarily dependent upon reduced ATP concentration or proton gradient potential, but rather upon the source of combined nitrogen. In this respect, incident light step down is not comparable with nutritional step-down procedures in heterotrophic bacteria.

Adenosine Triphosphate↗
PubMed1989

Correlation between histidine operon expression and guanosine 5'-diphosphate-3'-diphosphate levels during amino acid downshift in stringent and relaxed strains of Salmonella typhimurium.

We have analyzed the correlation of attenuator-independent expression of the Salmonella typhimurium histidine operon in vivo with levels of the "alarmone" guanosine 5'-diphosphate 3'-diphosphate. Amino acid downshift caused by serine hydroxamate addition increased his expression in a relA+ strain and decreased his expression in a relA mutant, whereas levels of guanosine 5'-diphosphate-3'-diphosphate varied in parallel with the changes in his expression in the two strains. In several experiments, overall variations in his expression ranged from 20- to 60-fold after downshift. The mild downshift allowed growth of the cultures to continue at near-preshift rates. Serine hydroxamate addition was also used to analyze the effect of amino acid downshift on induced expression of wild-type and mutant lac promoters. There was a 12-fold difference in lac expression when a relA+-relA1 pair was subjected to mild starvation but only a 3-fold difference when the strains carried the lacZpL8UV5 promoter mutation. These results suggest that guanosine 5'-diphosphate-3'-diphosphate stimulates gene expression in vivo at the level of transcription initiation.

Amino Acids↗
PubMed1976

Formation of uridine diphosphate 2-deoxy-D-glucose and guanosine diphosphate 2-deoxy-D-glucose in vitro using animal enzymes.

The enzymic formation of guanosine diphosphate 2-deoxy-D-glucose and uridine diphosphate 2-deoxy-D-glucose from synthetically prepared 2-deoxy-D-glucose 1-phosphate is described. Incubation of 2-deoxy-D-glucose 1-phosphate with an enzyme preparation from bovine mammary glands and either GTP or UTP gives rise to the corresponding nucleoside-diphosphate derivative of 2-deoxy-D-glucose. Uridine diphosphate 2-deoxy-D-glucose could also be obtained by incubation of 2-deoxy-D-glucose 1-phosphate with UTP and UDP glucose pyrophosphorylase from beef liver.

Animals↗
PubMed1996

Cyclic aristeromycin diphosphate ribose: a potent and poorly hydrolysable Ca(2+)-mobilising mimic of cyclic adenosine diphosphate ribose.

Cyclic aristeromycin diphosphate ribose, a carbocyclic analogue of cyclic adenosine diphosphate ribose, was synthesised using a chemo-enzymatic route involving activation of aristeromycin 5'-phosphate by diphenyl phosphochloridate. The calcium-releasing properties of this novel analogue were investigated in sea urchin egg homogenates. While cyclic aristeromycin diphosphate ribose has a calcium release profile similar to that of cyclic adenosine diphosphate ribose (EC50 values are 80 nM and 30 nM, respectively), it is degraded significantly more slowly (t1/2 values are 170 min and 15 min, respectively) and may, therefore, be a useful tool to investigate the activities of cyclic adenosine diphosphate ribose.

Adenosine↗
PubMed1987

2'-Deoxy-3'-O-(4-benzoylbenzoyl)- and 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate, fluorescent photoaffinity analogues of adenosine 5'-diphosphate. Synthesis, characterization, and interaction with myosin subfragment 1.

Two new fluorescent nucleotide photoaffinity labels, 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate (Bz2 epsilon ADP) and 2'-deoxy-3'-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate [3'(Bz2)2'd epsilon ADP], have been synthesized and used as probes of the ATP binding site of myosin subfragment 1 (SF1). These analogues are stably trapped by the bifunctional thiol cross-linker N,N'-p-phenylenedimaleimide (pPDM) at the active site in a manner similar to that of ATP [Wells, J.A., & Yount, R.G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970], and nonspecific photolabeling can be minimized by removing free probe by gel filtration prior to irradiation. Both probes covalently photoincorporate with high efficiency (40-50%) into the central 50-kDa heavy chain tryptic peptide, as found previously for the nonfluorescent parent compound 3'(2')-O-(4-benzoylbenzoyl)adenosine diphosphate [Mahmood, R., & Yount, R.G. (1984) J. Biol. Chem. 259, 12956-12959]. The solution conformations of Bz2 epsilon ADP and 3'(Bz2)-2'd epsilon ADP were analyzed by steady-state and time-resolved fluorescence spectroscopy. These data indicated that the benzoylbenzoyl rings in both analogues were stacked over the epsilon-adenine ring. The degree of stacking was greater with the 2' isomer than with the 3' isomer. Fluorescence quantum yields and lifetimes were measured for Bz2 epsilon ADP and 3'(Bz2)2'd epsilon ADP reversibly bound, stably trapped, and covalently photoincorporated at the active site of SF1. These values were compared with those for 3'(2')-O-[[(phenylhydroxymethyl)phenyl]carbonyl]-1,N6-ethenoadenos ine diphosphate (CBH epsilon ADP) and 2'-deoxy-3'-O-[[(phenylhydroxymethyl)phenyl]carbonyl]-1,N6- ethenoadenosine diphosphate [3'(CBH)2'd epsilon ADP]. These derivatives were synthesized as fluorescent analogues of the expected product of the photochemical reactions of Bz2 epsilon ADP and 3'(Bz2)2'd epsilon ADP, respectively, with the active site of SF1. The fluorescence properties of the carboxybenzhydrol derivatives trapped at the active site by pPDM were compared with those of the Bz2 nucleotide-SF1 complexes. These properties were consistent with a photoincorporation mechanism in which the carbonyl of benzophenone was converted to a tertiary alcohol attached covalently to the protein. The specific, highly efficient photoincorporation of Bz2 epsilon ADP at the active site will allow it to be used as a donor in distance measurements by fluorescence resonance energy transfer to acceptor sites on actin.

Adenosine Diphosphate↗
PubMed1976

Active site of ribonucleoside diphosphate reductase from Escherichia coli. Inactivation of the enzyme by 2'-substituted ribonucleoside diphosphates.

Ribonucleoside diphosphate reductase is an allosteric enzyme consisting of two nonidentical subunits, proteins B1 and B2. B1 contains dithiols which participate in the oxidation-reduction reactions of electron transport, while B2 contains a free radical essential for activity. Ribonucleoside diphosphates are bound to B1 but not to B2. Addition of 2'-deoxy-2'-chloro ribonucleoside diphosphates to ribonucleotide reductase irreversibly inactivates B1 without affecting B2. The reaction is specific since (a) it requires the presence of active B2, (b) it is controlled by allosteric effectors, (c) B1 is protected against inactivation by the normal substrates, and (d) the chloro-substituted nucleoside monophosphates have no effect. The inactivation of B1 is caused by a modification of the oxidation-reduction dithiols. The chloro derivatives decompose into free base, chloride ion, and 2-deoxyribose 5-diphosphate as a consequence of reaction with the enzyme. 2'-Deoxy-2"-azido ribonucleoside diphosphates cause an irreversible inactivation of B2 without affecting B1. The reaction is specific by the above criteria, indicating that the azido derivatives also bind to the active site of the enzyme. On reduction by ribonucleotide reductase, the azido derivatives function as radical scavengers and selectively destroy the free radical of B2, indicating that this radical participates in ribonucleotide reduction directly. On the basis of these results, a model of the active site of ribonucleotide reductase is proposed in which the site is formed from both B1 and B2. In the site the electron-donating oxidation-reduction active dithiols of B1 are in close contact with the free radical of B2. The azido derivative also inactivates the adenosylcobalamin-dependent ribonucleoside triphosphate reductase from Lactobacillus leichmannii and a ribonucleotide reductase preparation from calf thymus, indicating a general involvement of free radical intermediates in enzyme-catalyzed ribonucleotide reduction.

Escherichia coli↗
PubMed2002

Identification of a bisphosphonate that inhibits isopentenyl diphosphate isomerase and farnesyl diphosphate synthase.

We and others have recently shown that the major molecular target of nitrogen-containing bisphosphonate drugs is farnesyl diphosphate synthase, an enzyme in the mevalonate pathway. In an in vitro screen, we discovered a bisphosphonate, NE21650, that potently inhibited farnesyl diphosphate synthase but, unlike other N-BPs investigated, was also a weak inhibitor of isopentenyl diphosphate isomerase. NE21650 was a more potent inhibitor of protein prenylation in osteoclasts and macrophages, and a more potent inhibitor of bone resorption in vitro, than alendronate, despite very similar IC(50) values for inhibition of farnesyl diphosphate synthase. Our observations show that minor changes to the structure of bisphosphonates allow inhibition of more than one enzyme in the mevalonate pathway and suggest that loss of protein prenylation due to inhibition of more than one enzyme in the mevalonate pathway may lead to an increase in antiresorptive potency compared to bisphosphonates that only inhibit farnesyl diphosphate synthase.

Alendronate↗
PubMed1988

Determination of isopentenyl diphosphate and farnesyl diphosphate in tissue samples with a comment on secondary regulation of polyisoprenoid biosynthesis.

A double-isotope dilution procedure is described for the determination of two isoprenoid precursors, isopentenyl and farnesyl diphosphate. Recovery of each is determined by the addition of the appropriate radioactive diphosphate to the tissue sample. After partial purification, each is coupled by a prenyltransferase with a cosubstrate of known specific activity. The products, doubly labeled farnesyl and geranylgeranyl diphosphates, are cleaved to the parent alcohols by alkaline phosphatase. The resulting polyprenols are isolated by reversed-phase thin-layer chromatography and their radioisotopic content is determined. The levels of these precursors have been measured in livers of rats and mice that have been maintained on several different diets. The concentration of each was about 0.5 mumol/g wet tissue and varied as much as 10-fold under the different test conditions. The levels of isopentenyl diphosphate isomerase, farnesyl diphosphate synthetase, and squalene synthetase were also measured in these animals. The changes in levels of these enzymes, in conjunction with the variation in substrate concentrations, are such that they could substantially influence the rate of cholesterol synthesis in liver.

Animals↗