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At least 19 recordsLinked to original sources
PubMed1992

Farnesyl diphosphate synthase and solanesyl diphosphate synthase reactions of diphosphate-modified allylic analogs: the significance of the diphosphate linkage involved in the allylic substrates for prenyltransferase.

Diphosphate-modified substrates for prenyltransferase were synthesized and examined as substrates for the prenyltransferase reaction. They were dimethylallyl methylenediphosphonate, geranyl methylenediphosphonate, geranyl imidodiphosphate, geranyl phosphosulfate, farnesyl methylenediphosphonate, farnesyl imidodiphosphate, and farnesyl phosphosulfate. All of them except dimethylallyl methylenediphosphonate were accepted as substrates by solanesyl diphosphate synthase to give solanesyl diphosphate and the former four analogs were also accepted as substrates by farnesyl diphosphate synthase to give farnesyl diphosphate. The Km values of both enzymes for the methylenediphosphonate and imidodiphosphate analogs were comparable to those of the corresponding diphosphate substrates, but the phosphosulfate analogs showed much greater Km values than the diphosphate substrates. On the other hand, the Vmax values for these artificial substrates were all smaller than those for the corresponding natural substrates. Kinetic experiments with the analogs showed that the ionization-condensation-elimination mechanism proposed for the farnesyl diphosphate synthase reaction holds also for the solanesyl diphosphate synthase reaction and that the diphosphoryl structure, capable of chelating with divalent cations, is important topologically and kinetically rather than thermodynamically.

Alkyl and Aryl Transferases↗
PubMed2001

Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate.

Geranyl diphosphate synthase belongs to a subgroup of prenyltransferases, including farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, that catalyzes the specific formation, from C(5) units, of the respective C(10), C(15), and C(20) precursors of monoterpenes, sesquiterpenes, and diterpenes. Unlike farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, which are homodimers, geranyl diphosphate synthase from Mentha is a heterotetramer in which the large subunit shares functional motifs and a high level of amino acid sequence identity (56-75%) with geranylgeranyl diphosphate synthases of plant origin. The small subunit, however, shares little sequence identity with other isoprenyl diphosphate synthases; yet it is absolutely required for geranyl diphosphate synthase catalysis. Coexpression in Escherichia coli of the Mentha geranyl diphosphate synthase small subunit with the phylogenetically distant geranylgeranyl diphosphate synthases from Taxus canadensis and Abies grandis yielded a functional hybrid heterodimer that generated geranyl diphosphate as product in each case. These results indicate that the geranyl diphosphate synthase small subunit is capable of modifying the chain length specificity of geranylgeranyl diphosphate synthase (but not, apparently, farnesyl diphosphate synthase) to favor the production of C(10) chains. Comparison of the kinetic behavior of the parent prenyltransferases with that of the hybrid enzyme revealed that the hybrid possesses characteristics of both geranyl diphosphate synthase and geranylgeranyl diphosphate synthase.

Alkyl and Aryl Transferases↗
PubMed1993

Geranylgeranyl diphosphate synthase catalyzing the single condensation between isopentenyl diphosphate and farnesyl diphosphate.

Geranylgeranyl diphosphate synthase was purified 191-fold from bovine brain by Mono Q column chromatography followed by preparative isoelectric focusing electrophoresis and Superose 12 gel filtration. The synthase had a pI value at 6.0, and it was made free of farnesyl diphosphate synthase, the pI of which was 5.1. The partially purified enzyme catalyzed the formation of geranylgeranyl diphosphate from isopentenyl diphosphate and farnesyl diphosphate with the Km values for isopentenyl diphosphate and farnesyl diphosphate being 14 and 0.8 microM, respectively. Dimethylallyl diphosphate and geranyl diphosphate were poor substrates with velocities of only 0.003 and 0.03, respectively, relative to that of farnesyl diphosphate. These results indicate that geranylgeranyl diphosphate synthase catalyzes a single condensation between isopentenyl diphosphate and farnesyl diphosphate and that farnesyl diphosphate is the common intermediate at the branch point for the synthesis of geranylgeranylated proteins as well as cholesterol, ubiquinone, dolichol, and farnesylated proteins. The enzyme required Mg2+ or Mn2+ for maximum activity. Octylglucoside showed a stimulatory effect on the enzyme activity.

Alkyl and Aryl Transferases↗
PubMed1999

Acid-Base and Metal Ion-Coordinating Properties of Pyrimidine-Nucleoside 5'-Diphosphates (CDP, UDP, dTDP) and of Several Simple Diphosphate Monoesters. Establishment of Relations between Complex Stability and Diphosphate Basicity.

The stability constants of the 1:1 complexes formed between Mg(2+), Ca(2+), Sr(2+), Ba(2+), Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), or Cd(2+) and the pyrimidine-nucleoside 5'-diphosphates CDP(3)(-), UDP(3)(-), and dTDP(3)(-) (= NDP(3)(-)) were determined by potentiometric pH titration in aqueous solution (I = 0.1 M, NaNO(3); 25 degrees C). For comparison, the same values were measured for the corresponding complexes with the simple diphosphate monoesters (R-DP(3)(-)) phenyl diphosphate, methyl diphosphate, and n-butyl diphosphate. The acidity constants for H(3)(CDP)(+/-), H(2)(UDP)(-), H(2)(dTDP)(-), and H(2)(R-DP)(-) were measured also via potentiometric pH titration and various comparisons with related constants are made. By plotting log versus for the complexes of all six diphosphates mentioned and by a careful evaluation of the deviation of the various data pairs from the straight-line correlations, the expectation is confirmed that in the M(UDP)(-) and M(dTDP)(-) complexes the metal ion is only diphosphate-coordinated. The straight-line equations, which result from the mentioned correlations, together with the pK(a) value of a given monoprotonated diphosphate monoester allow now to predict the stability of the corresponding M(R-DP)(-) complexes. In this way, the experimentally determined stability constants for the M(CDP)(-) complexes are evaluated and it is concluded that the pyridine-like N3 of the cytosine residue does not participate in complex formation; i.e., the stability of the M(CDP)(-) complexes is also solely determined by the coordination tendency of the diphosphate residue. In all the monoprotonated M(H;NDP) and M(H;R-DP) complexes both, H(+) and M(2+), are bound at the diphosphate group. Only the Cu(H;CDP) complex exists in aqueous solution in the form of three different isomers: about 15% of the species have Cu(2+) and H(+) at the diphosphate residue, in about 13% Cu(2+) is bound at N3 and H(+) at the terminal beta-phosphate group, and the dominating isomer with about 72% carries the proton at N3 and the metal ion at the diphosphate residue. Several general features of phosphate-metal ion coordination are discussed, and estimations for the stabilities of the Fe(2+) complexes formed with mono-, di-, and triphosphate monoesters are provided.

Journal Article↗
PubMed1975

Nucleotide specificity of stringent factor and the synthesis of analogs of guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate.

The ribosome-dependent stringent factor reaction was found to be nonspecific with regard to the number of phosphate groups linked to the 5' position of guanosine nucleotides. Both GMP and guanosine 5'-tetraphosphate could accept a pyrophosphoryl group from ATP although at a much lower rate than GDP or GTP. Guanosine 5'-monophosphate 3'-diphosphate and guanosine 5'-tetraphosphate 3'-diphosphate were the products of these reactions. Furthermore, 3'-linked analogs of guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate were synthesized from the corresponding ATP analog, adenosine 5'-O-(3-thiotriphosphate). The stringent factor catalyzed reverse reaction was found to be specific for guanosine 5'-diphosphate 3'-diphosphate, and was essentially inactive to the isomeric form, guanosine 5'-diphosphate 2'-diphosphate.

Bacterial Proteins↗
PubMed1972

Purification and properties of the adenosine diphosphate-glucose and uridine diphosphate-glucose pyrophosphorylases of Mycobacterium smegmatis: inhibition and activation of the adenosine diphosphate-glucose pyrophosphorylase.

Crude extracts of Mycobacterium smegmatis catalyzed the synthesis of adenosine diphosphate-glucose (ADP-Glc), cytidine diphosphate-glucose, guanosine diphosphate-glucose (GDP-Glc), thymidine diphosphate-glucose (TDP-Glc), and uridine diphosphate-glucose (UDP-Glc). In these crude enzyme fractions, high concentrations of trehalose-P inhibited the ADP-Glc and GDP-Glc pyrophosphorylases but did not effect the UDP-Glc or TDP-Glc pyrophosphorylases. Both the ADP-Glc pyrophosphorylase and the UDP-Glc pyrophosphorylase were partially purified (about 140-fold and 60-fold, respectively), and their properties were compared. For the ADP-Glc pyrophosphorylase, the K(m) for adenosine triphosphate was 6 x 10(-4)m, whereas that for glucose-1-P was 8 x 10(-4)m. The optimal concentration of Mg(2+) was 1 x 10(-3)m, and the pH optimum was 8.5. For the UDP-Glc pyrophosphorylase, the K(m) for uridine triphosphate was 1 x 10(-3)m and for glucose-1-P was 2 x 10(-3)m. The optimal Mg(2+) concentration was 1 x 10(-3)m, and the pH optimum was about 8.0. The purified ADP-Glc pyrophosphorylase was inhibited by fructose-6-P, fructose-1, 6-diphosphate, glucose-6-P, and phosphoenolpyruvate. On the other hand, trehalose, trehalose diphosphate, sodium pyruvate, and ribose-5-P did not effect the ADP-Glc pyrophosphorylase. None of these compounds, including trehalose-P, had any effect on the UDP-Glc pyrophosphorylase.

Adenosine↗
PubMed1978

Intracellular levels of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) in cultures of Streptomyces griseus producing streptomycin.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) were identified in the vegative mycelium of Streptomyces griseus. Adenosine 5'-diphosphate 3'-diphosphate (ppApp) and adenosine 5'-triphosphate 3'-diphosphate (pppApp) were not present but several other phosphorus-containing compounds which may have been inorganic polyphosphates were detected. During exponential growth of S. griseus the concentrations of ppGpp and pppGpp were several times higher than in the stationary stage. They fell sharply when exponential growth ended and then remained at an almost constant basal level. For the tetraphosphate the maximum concentration was about 50, and for the basal level about 10, pmol per millilitre of a culture with an optical density of 1.0. Production of streptomycin started several hours after exponential growth had ended and the concentrations of ppGpp and pppGpp had fallen. Streptomycin synthesis was delayed if the cells were resuspended just before production started in fresh medium lacking phosphate, but it was not delayed by glucose starvation. Both cultures, as well as cultures transferred to nitrogen-free medium, showed an immediate increase in ppGpp content to about four-fold the basal level. The results suggest that the guanosine polyphosphates do not directly control initiation of streptomycin production in S. griseus. Twelve additional species of Streptomyces examined all contained ppGpp and pppGpp.

Culture Media↗
PubMed1982

Competitive inhibition by adenosine 5'-triphosphate of the actions on human platelets of 2-chloroadenosine 5'-diphosphate, 2-azidoadenosine 5'-diphosphate and 2-methylthioadenosine 5'-diphosphate.

1 Adenosine 5'-diphosphate (ADP) induces human platelet aggregation and noncompetitively inhibits stimulated human platelet adenylate cyclase; these two effects are mediated by the same ADP receptor, at which adenosine 5'-triphosphate (ATP) is a competitive antagonist. 2 Two ADP analogues, 2-azidoadenosine 5'-diphosphate (2-azido-ADP) and 2-methylthioadenosine 5'-diphosphate (2-methylthio-ADP) have been reported to be more potent as inhibitors of adenylate cyclase than they are as aggregating agents, but no evidence has been presented that these actions are mediated solely by the ADP receptor. 3 We therefore tested the ability of ATP to inhibit the actions of these compounds and of another ADP analogue, 2-chloroadenosine 5'-diphosphate (2-chloro-ADP). 4 2-Chloro-ADP, 2-azido-ADP and 2-methylthio-ADP each induced aggregation and inhibited stimulated adenylate cyclase. Both of these actions were competitively inhibited by ATP (50 microM) with pA2 values similar to those previously found for inhibition by ATP of these effects of ADP. 5 The reported greater potency of 2-azido-ADP and of 2-methylthio-ADP as inhibitors of adenylate cyclase than as aggregating agents is therefore due only to their greater efficacy for this effect, not to some extra actions elsewhere.

Adenosine Diphosphate↗
PubMed1997

Conversion from archaeal geranylgeranyl diphosphate synthase to farnesyl diphosphate synthase. Two amino acids before the first aspartate-rich motif solely determine eukaryotic farnesyl diphosphate synthase activity.

Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are precursors for a variety of important natural products, such as sterols, carotenoids, and prenyl quinones. Although FPP synthase and GGPP synthase catalyze similar consecutive condensations of isopentenyl diphosphate with allylic diphosphates and have several homologous regions in their amino acid sequences, nothing is known about how these enzymes form the specific products. To locate the region that causes the difference of final products between GGPP synthase and FPP synthase, we constructed six mutated archaeal GGPP synthases whose regions around the first aspartate-rich motif were replaced with the corresponding regions of FPP synthases from human, rat, Arabidopsis thaliana, Saccharomyces cerevisiae, Escherichia coli, Bacillus stearothermophilus, and from some other related mutated enzymes. From the analysis of these mutated enzymes, we revealed that the region around the first aspartate-rich motif is essential for the product specificity of all FPP synthases and that the mechanism of the chain termination in eukaryotic FPP synthases (type I) is different from those of prokaryotic FPP synthases (type II). In FPP synthases of type I, two amino acids situated at the fourth and the fifth positions before the motif solely determine their product chain length, while the product specificity of the type II enzymes is determined by one aromatic amino acid at the fifth position before the motif, two amino acids inserted in the motif, and other modifications. These data indicate that FPP synthases have evolved from the progenitor corresponding to the archaeal GGPP synthase in two ways.

Alkyl and Aryl Transferases↗
PubMed1980

A radioimmunoassay for guanosine-5'-diphosphate-3'-diphosphate and adenosine-5'-triphosphate-3'-diphosphate.

A radioimmunoassay for guanosine-5'-diphosphate-3'-diphosphate (ppGpp) and adenosine-5'-triphosphate-3'-diphosphate (pppApp) has been developed. The assay method is based on competition of an unlabeled highly phosphorylated nucleotide with 3H-labeled highly phosphorylated nucleotide for binding sites on a specific antibody. Antibodies to ppGpp and pppApp were obtained by immunizing rabbits with the antigen prepared by conjugating ppGpp with human serum albumin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and with the antigen prepared by conjugating 8-(6-aminohexyl)amino-adenosine-5'-triphosphate-3'-diphosphate with human serum albumin using glutaraldehyde, respectively. Antibody-bound 3H-labeled highly phosphorylated nucleotides were separated from the free 3H-labeled highly phosphorylated nucleotides by selective adsorption on dextran-coated charcoal. Displacement plots were linear over a concentration range of 5-1,000 pmol/assay tube in a log-probit percentage plot. Application of this method to biological systems offers improved accuracy and convenience compared with the previous 32PO4-labeling technique.

Adenine Nucleotides↗
PubMed1978

Characterization of the guanosine 5'-triphosphate 3'-diphosphate and guanosine 5'-diphosphate 3'-diphosphate degradation reaction catalyzed by a specific pyrophosphorylase from Escherichia coli.

Guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and guanosine 5'-diphosphate 3'-diphosphate (ppGpp) are specifically degraded by a manganese-dependent pyrophosphorylase present in spoT+ but not in spoT- strains of Escherichia coli, indicating that the enzyme is the spoT gene product. The enzyme catalyzes the release of pyrophosphate from the 3' position of ppGpp or pppGpp, yielding ppG and pppG, respectively; pppGpp could not be detected as an intermediate in the decay reaction. Degradation of (p)ppGpp is optimal in the presence of 200 to 300 mM potassium or sodium acetate, at a pH of 7.5 to 8 and a temperature of 37 degrees C.

Escherichia coli↗
PubMed2005

Isopentenyl diphosphate isomerase. Mechanism-based inhibition by diene analogues of isopentenyl diphosphate and dimethylallyl diphosphate.

Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This is an essential step in the mevalonate entry into the isoprenoid biosynthetic pathway. The isomerization catalyzed by type I IDI involves protonation of the carbon-carbon double bond in IPP or DMAPP to form a tertiary carbocation, followed by deprotonation. Diene analogues for DMAPP (E-2-OPP and Z-2-OPP) and IPP (4-OPP) were synthesized and found to be potent active-site-directed irreversible inhibitors of the enzyme. X-ray analysis of the E.I complex between Escherichia coli IDI and 4-OPP reveals the presence of two isomers that differ in the stereochemistry of the newly formed C3-C4 double bond in the hydrocarbon chain of the inhibitor. In both adducts C5 of the inhibitor is joined to the sulfur of C67. In these structures the methyl group formed upon protonation of the diene moiety in 4-OPP is located near E116, implicating that residue in the protonation step.

Carbon-Carbon Double Bond Isomerases↗
PubMed2003

Changes in the concentrations of guanosine 5'-diphosphate 3'-diphosphate and the initiating nucleoside triphosphate account for inhibition of rRNA transcription in fructose-1,6-diphosphate aldolase (fda) mutants.

Early screens for conditional lethal mutations that affected rRNA expression in Escherichia coli identified temperature-sensitive fda mutants (fda encodes the glycolytic enzyme fructose-1,6-diphosphate aldolase). It was shown that these fda(Ts) mutants were severely impaired in rRNA synthesis upon shift to the restrictive temperature, although the mechanism of inhibition was never determined. Here, we bring resolution to this long-standing question by showing that changes in the concentrations of guanosine 5'-diphosphate 3'-diphosphate and initiating nucleoside triphosphates can account for the previously observed effects of fda mutations on rRNA transcription.

Adenosine Triphosphate↗
PubMed1992

Studies on geranylgeranyl diphosphate synthase from rat liver: specific inhibition by 3-azageranylgeranyl diphosphate.

Geranylgeranyl diphosphate synthase from rat liver was separated from farnesyl diphosphate synthase, the most abundant and widely occurring prenyltransferase, by DEAE-Toyopearl column chromatography. The enzyme catalyzed the formation of E,E,E-geranylgeranyl diphosphate (V) from isopentenyl diphosphate (II) and dimethylallyl diphosphate (I), geranyl diphosphate (III), or farnesyl diphosphate (IV) with relative velocities of 0.09:0.15:1. 3-Azageranylgeranyl diphosphate (VII), designed as a transition-state analog for the geranylgeranyl diphosphate synthase reaction, was synthesized and found to act as a specific inhibitor for this synthase, but not for farnesyl diphosphate synthase. Diphosphate V and its Z,E,E-isomer (VI) also inhibited geranylgeranyl diphosphate synthase, but the effect was not as striking as that of the aza analog VII. Specific inhibition of geranylgeranyl diphosphate synthase by VII was also observed in experiments with 100,000g supernatants of rat brain and liver homogenates which contained isopentenyl diphosphate isomerase and prenyltransferases including farnesyl diphosphate synthase as well as geranylgeranyl diphosphate synthase. For farnesyl:protein transferase from rat brain, however, the aza compound did not show a stronger inhibitory effect than E,E,E-geranylgeranyl diphosphate.

Animals↗
PubMed2000

Identification of a short (C15) chain Z-isoprenyl diphosphate synthase and a homologous long (C50) chain isoprenyl diphosphate synthase in Mycobacterium tuberculosis.

We report the cloning, overexpression, and partial characterization of two unique Z-isoprenyl diphosphate synthase homologs from Mycobacterium tuberculosis. The first enzyme, Rv1086, adds one isoprene unit to omega,E-geranyl diphosphate. The product, omega,E, Z-farnesyl diphosphate, is the putative substrate of the second enzyme, Rv2361c. This enzyme adds seven more isoprene units to omega, E,Z-farnesyl diphosphate and releases decaprenyl diphosphate. Both open reading frames were cloned from the M. tuberculosis H37Rv genome and overexpressed in M. smegmatis. Membrane and cytosol fractions from wild type and the two recombinant strains were assayed for [(14)C]isopentenyl diphosphate incorporation into isoprenyl diphosphates in the presence of various allylic isoprenyl diphosphate acceptors. Membrane fractions of recombinant cells overexpressing Rv2361c incubated with farnesyl diphosphate showed a 10-fold increase of [(14)C]isopentenyl diphosphate incorporation into decaprenyl diphosphate. Membrane fractions of recombinant cells overexpressing Rv1086 incubated with geranyl diphosphate showed a 5-fold increase of [(14)C]isopentenyl diphosphate incorporation into farnesyl diphosphate. Analysis of the stereochemistry revealed that all of the overexpressed farnesyl diphosphate was in the omega,E, Z-configuration. This is the first description of a short chain isoprenyl diphosphate synthase that generates products with Z-stereochemistry. Previously, all known short chain isoprenyl diphosphate synthases catalyze the synthesis of products with E-stereochemistry.

Alkyl and Aryl Transferases↗