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PubMedDate not supplied

Synthesis, cytostatic activity and inhibition of ribonucleotide reductase by 5'-phosphoramidates and 5'-diphosphates, of 2'-O-allyl-arabinofuranosyl nucleosides.

The diphosphates of a series of 2'-O-allyl-1-beta-D-arabinofuranosyl derivatives, previously obtained by us, have been prepared and tested for their inhibitory activity in an in vitro assay using R1 and R2 subunits of the purified recombinant mouse ribonucleotide reductase (RNR). 2'-O-Allyl-araU diphosphate proved to be inhibitory, with an IC50 of 100 microM. The 5'-phosphoramidate pronucleotide of 2'-O-allyl-araU was also prepared and tested for inhibition of tumor cell proliferation.

Animals↗
PubMed1970

Chloroplast structure and function in ac-20, a mutant strain of Chlamydomonas reinhardi. I. CO2 fixation and ribulose-1,5-diphosphate carboxylase synthesis.

A mutant strain of the green alga Chlamydomonas reinhardi, ac-20, is described in which both the rate of CO(2) fixation by whole cells and the rate of carboxylation of ribulose-1,5-diphosphate in cell-free extracts are reduced, particularly when sodium acetate is present in the growth medium. Of the enzymes of the reductive pentose phosphate cycle tested, only ribulose-1,5-diphosphate carboxylase activity is reduced in the mutant strain, and it appears that the low carboxylase activity limits the strain's rate of photosynthetic carbon metabolism. Evidence is presented to show that the fluctuation in the level of the enzyme activity in the presence or absence of acetate results from the fluctuation in the level of some factor(s) limiting the rate of synthesis of the protein.

Acetates↗
PubMed2006

Leigh syndrome with nephropathy and CoQ10 deficiency due to decaprenyl diphosphate synthase subunit 2 (PDSS2) mutations.

Coenzyme Q(10) (CoQ(10)) is a vital lipophilic molecule that transfers electrons from mitochondrial respiratory chain complexes I and II to complex III. Deficiency of CoQ(10) has been associated with diverse clinical phenotypes, but, in most patients, the molecular cause is unknown. The first defect in a CoQ(10) biosynthetic gene, COQ2, was identified in a child with encephalomyopathy and nephrotic syndrome and in a younger sibling with only nephropathy. Here, we describe an infant with severe Leigh syndrome, nephrotic syndrome, and CoQ(10) deficiency in muscle and fibroblasts and compound heterozygous mutations in the PDSS2 gene, which encodes a subunit of decaprenyl diphosphate synthase, the first enzyme of the CoQ(10) biosynthetic pathway. Biochemical assays with radiolabeled substrates indicated a severe defect in decaprenyl diphosphate synthase in the patient's fibroblasts. This is the first description of pathogenic mutations in PDSS2 and confirms the molecular and clinical heterogeneity of primary CoQ(10) deficiency.

Alkyl and Aryl Transferases↗
PubMed2004

Relationship between intron 4b splicing of the rat geranylgeranyl diphosphate synthase gene and the active enzyme expression level.

Geranylgeranyl diphosphate synthase (GGPS) is a branch point enzyme in the mevalonate pathway that catalyzes the synthesis of geranylgeranyl diphosphate used for the geranylgeranylation of Rho, Rac and Rab proteins. The current study showed the production of multiple forms of GGPS mRNA from a single GGPS gene in rat. The mRNAs resulted from combinations of multiple alternative introns and two poly(A) sites in the 3'-translated and 3'-untranslated regions. These are classified into 1a-type and 1b-type mRNAs, based on the splicing of intron 4b resulting in the difference in deduced amino acid sequence between the C-terminal regions. The 1a-type and 1b-type proteins expressed in both Escherichia coli and HeLa cells were active and inactive, respectively. In the case of HeLa cells, the latter protein expression level was about 10% relative to the former one. This was also observed for Cos-7 and 293 cells. When fusions of beta-galactosidase with C-terminal regions differing between the 1a-type and 1b-type proteins were expressed in HeLa cells, the expressed fusion proteins were both found to be active but the latter fusion protein expression level was considerably low compared with the former one. The expression level of 1a-type mRNA was higher than that of 1b-type mRNA in brain, liver, heart, and thymus, but the two expression levels were the same in testis and ovary. During testis development the total GGPS mRNA expression level increased, accompanied by an increase in 1b-type mRNA, the expression level of 1a-type mRNA encoding active GGPS remaining kept unchanged. These results indicate that the expression level of rat active GGPS is at least regulated through the splicing of intron 4b of its gene.

3' Untranslated Regions↗
PubMed1978

Enzymatic synthesis of oligodeoxyribonucleotides of defined sequence. Polynucleotide phosphorylase catalysed synthesis using pyrimidine analog-containing deoxyribonucleoside 5'-diphosphates.

The E. coli polynucleotide phosphorylase-catalysed reaction of the deoxynucleoside 5'-diphosphates of 5-methyldeoxycytidine, N4-hydroxydeoxycytidine, deoxyuridine and 5-mercurideoxyuridine with the primers d(pT-T-A-G) and d(pT-T-T-T-T-T) have been studied under conditions where the primer is extended, predominantly, by one or two nucleotide residues. In experiments with 5-mercurideoxyuridine 5'-diphosphate, no 5-mercurideoxy-uridine-containing oligonucleotides were produced. The other three nucleotide analogs were found to be good substrates for E. coli PNPase and the conditions established for synthesis with these analogs will allow the construction of a number of biologically useful types of oligodeoxyribonucleotide.

Chromatography, High Pressure Liquid↗
PubMed1979

Synthesis and coding properties of dinucleoside diphosphates containing alky pyrimidines which are formed by the action of carcinogens on nucleic acids.

Dinucleoside diphosphates of the general type pGpN have been prepared enzymatically using ribonuclease N1. Alkylated uridines or cytidines, which are products of carcinogens acting on nucleic acids, were tested in dinucleoside diphosphates for their ability to stimulate the binding of Ala- or Val-tRNA to ribosomes. O2-Ethyl C and 3-methyl C functioned as U, but not as C. In contrast, 3-methyl U behaved as C, but not as U. Both O2 and O4-ethyl U could be recognized as C or U, although binding in both cases was weak. Thus, modifications of the hydrogen-bonding sites of U or C causes miscoding and could be considered to represent mutagenic reactions.

Codon↗
PubMed1988

Undecaprenyl diphosphate synthase from Micrococcus luteus B-P 26: essential factors for the enzymatic activity.

An undecaprenyl diphosphate synthase fraction, which was free of other prenyltransferases and was active without the addition of detergent or phospholipid, was obtained by Sephadex G-100 chromatography of cell-free extracts of Micrococcus luteus B-P 26 cells. The addition of small amounts of Triton X-100 to this fraction caused a marked loss of the enzyme activity, but the activity was gradually restored as further detergent was added. When the enzyme fraction was chromatographed on DEAE-cellulose, the synthase was partially purified, but the activity was not detected unless assayed with addition of the detergent or a lipid fraction of this bacterium. Among the three phospholipids isolated from this bacterium, cardiolipin and phosphatidylglycerol had a marked effect in activating lipid-depleted undecaprenyl diphosphate synthase, but O-lysylphosphatidylglycerol, which occurs prominently in this bacterium, had little effect.

Alkyl and Aryl Transferases↗
PubMed1993

Solubilization and characterization of dehydrodolichyl diphosphate synthase from the yeast Saccharomyces carlsbergensis.

When the membrane fraction of Saccharomyces carlsbergensis was incubated with radiolabeled isopentenyl diphosphate in the presence of farnesyl diphosphate and Mg2+, phosphorylated and free long-chain polyprenols were formed. The reaction was inhibited by EDTA and heavy metal cations. A series of non-ionic detergents were studied for their efficacy to solubilize the prenyltransferase. The enzyme completely lost its activity in the presence of 0.1% of Triton X-100. n-Octyl-beta-(D)glucopyranoside at the concentration of 0.25-0.5% (10-15 mM) was used to solubilize the prenyltransferase. Both the membrane-bound enzyme and the solubilizate possessed a broad pH optimum shifted to alkaline pH values. The temperature optimum of the solubilizate was somewhat lower than that of the membrane preparation, owing to the significantly lower thermostability of the solubilized enzyme in comparison with the membrane-bound one. The phosphorylated reaction products formed in the presence of the membrane preparation had the same composition as the yeast dolichol synthesized in vivo. Non-phosphorylated polyprenols were formed during the incubation with membranes but not the solubilized enzyme. The composition of the polyprenols was also coincident with that of yeast dolichol, and the individual C80-homolog of the mixture was polyprenol but not dolichol as judged by adsorption HPLC. The results are discussed in relation to the terminal stages in the biosynthesis of dolichol derivatives.

Alkyl and Aryl Transferases↗
PubMed1975

Fructose-diphosphate aldolase of Horseshoe crab (Tachypleus tridentatus).

Fructose-diphosphate aldolase [ED 4.1.2.13] was isolated from horseshoe crab ( living fossil) muscle and some molecular and enzymatic properties were examined. The enzyme was a tetramer with a molecular weight of about 160,000. The enzyme activity was inhibited by reduction with borohydride in the presence of the substrate and was inactivated by carboxypeptidase A [EC 3.4.12.2] digestion. The pH optima for fructose-diphosphate (FDP) and fructose-1-phosphate (F1P) activities were 6.5--8 and 7.5--8.2, respectively. The ratio of FDP/F1P activities was 30 and Km values were 1.7 times 10- minus 5 M and 2.5 times 10- minus 3 M, respectively, for the two substrates. The horseshoe crab aldolase was classified as class 1, type A, based on the results obtained. Extensive homology in various properties of the enzyme was observed when it was compared with enzymes from other sources, though some differences could be found in the amino acid composition and in the kinetic properties.

Amino Acids↗
PubMed1975

Novel monofunctional substrates of polynucleotide phosphorylase. The "single-addition" of 2'(3')-O-dihydrocinnamoyl-nucleoside 5'-diphosphate to a primer oligonucleotide.

A method was developed for stepwise wynthesis of oligonucleotides of difined wequence using 2'(3')-O-dihydrocinnamoyl-nucleoside 5'-diphosphates as substrates for polynucleotide phosphorylase [ED 2.7.7.8]. Polynucleotide phosphorylase from Thermus thermophilus catalyzed the transfer of one 2'(3')-blocked ADP to the 3'-terminus of the primer trinucleoside diphosphate, ApApA. The product was 2'(3')-substituted triadenylyladenosine. The blocking group, dihydrocinnamoyl, could be removed completely from the product without destruction of the phosphodiester bond using alpha-chymotrypsin [ED 3.4.21.1] at neutral pH.

Bacteria↗
PubMed1976

Enzymatic synthesis of oligonucleotides of defined sequence. The "single addition" of 2(3)-O-dihydrocinnamoyl-nucleoside 5'-diphosphate to a primer oligonucleotide catalyzed by a thermophilic polynucleotide phosphorylase.

Several oligonucleotides of defined sequence were synthesized using 2'(3')-O-dihydrocinnamoyl-nucleoside 5'-diphosphates (DHC-NDP) as substrates for polynucleotide phosphorylase [EC 2.7.7.8] from Thermus thermophilus. The enzyme catalyzed the transfer of one nucleotidyl residue from each of the 2'(3')-O-dihydrocinnamoyl esters of CDP, UDP, and GDP to the 3'-terminus of the primer triadenosine diphosphate, (Ap)2A. The products were shown to be (Ap)3C, (Ap)3U, and (Ap)3G by enzymatic analysis.

Adenine Nucleotides↗
PubMed1993

Identification of the uridine-binding domain of sucrose-phosphate synthase. Expression of a region of the protein that photoaffinity labels with 5-azidouridine diphosphate-glucose.

The uridine diphosphate-glucose (UDP-Glc) binding domain of sucrose-phosphate synthase (SPS) was identified by overexpressing part of the gene from spinach (Spinacia oleracea). Degenerate oligonucleotide primers corresponding to two tryptic peptides common to both the full-length 120-kD SPS subunit and an 82-kD form that photoaffinity labeled with 5-azidouridine diphosphate-glucose (5-N3UDP-Glc) were used in a polymerase chain reaction to isolate a partial cDNA clone. Comparison of the deduced amino acid sequence of spinach SPS with the sequences of potato sucrose synthase showed that the partial cDNA included one region that was highly conserved between the proteins. Expression of the partial cDNA clone of SPS in Escherichia coli produced a 26-kD fusion protein that photoaffinity labeled with 5-N3UDP-Glc. Photoaffinity labeling of the 26-kD fusion protein was specific, indicating that this portion of the SPS protein harbors the UDP-Glc-binding domain. Isolation of a modified peptide from the photolabeled protein provided tentative identification of amino acid residues that make up the uridine-binding domain of SPS.

Amino Acid Sequence↗
PubMed2004

Organization of monoterpene biosynthesis in Mentha. Immunocytochemical localizations of geranyl diphosphate synthase, limonene-6-hydroxylase, isopiperitenol dehydrogenase, and pulegone reductase.

We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (-)-(4S)-limonene-6-hydroxylase, peppermint (-)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (-)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (-)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity.

Alcohol Oxidoreductases↗
PubMed2004

Rice contains two disparate ent-copalyl diphosphate synthases with distinct metabolic functions.

Rice (Oryza sativa) produces ent-copalyl diphosphate for both gibberellin (GA) phytohormone and defensive phytoalexin biosynthesis, raising the question of how this initial biosynthetic step is carried out for these distinct metabolic processes. Here, a functional genomics approach has been utilized to identify two disparate ent-copalyl diphosphate synthases from rice (OsCPS1ent and OsCPS2ent). Notably, it was very recently demonstrated that only one of these (OsCPS1ent) normally operates in GA biosynthesis as mutations in this gene result in severely impaired growth. Evidence is presented here strongly indicating that the other (OsCPS2ent) is involved in related secondary metabolism producing defensive phytochemicals. In particular, under appropriate conditions, OsCPS2ent mRNA is specifically induced in leaves prior to production of the corresponding phytoalexins. Thus, transcriptional control of OsCPS2ent seems to be an important means of regulating defensive phytochemical biosynthesis. Finally, OsCPS1ent is significantly more similar to the likewise GA-specific gene An1/ZmCPS1ent in maize (Zea mays) than its class II terpene synthase paralogs involved in rice secondary metabolism. Hence, we speculate that this cross-species conservation by biosynthetic process reflects derivation of related secondary metabolism from the GA primary biosynthetic pathway prior to the early divergence between the separate lineages within the cereal/grass family (Poaceae) resulting in modern rice and maize.

Alkyl and Aryl Transferases↗
PubMed1995

Adenosine 5'-diphosphate-glucose pyrophosphorylase from potato tuber. Significance of the N terminus of the small subunit for catalytic properties and heat stability.

cDNAs encoding the large subunit and a possibly truncated small subunit of the potato tuber (Solanum tuberosum L.) adenosine 5'-diphosphate-glucose pyrophosphorylase have been expressed in Escherichia coli (A.A. Iglesias, G.F. Barry, C. Meyer, L. Bloksberg, P.A. Nakata, T. Greene, M.J. Laughlin, T.W. Okita, G.M. Kishore, J. Preiss, J Biol Chem [1993] 268: 1081-1086). However, some properties of the transgenic enzyme were different from those reported for the enzyme from potato tuber. In this work, extension of the cDNA was performed to elongate the N terminus of the truncated small subunit by 10 amino acids. This extension is based on the almost complete conservation seen at the N-terminal sequence for the potato tuber and the spinach leaf small subunits. Expressing the extended cDNA in E. coli along with the large subunit cDNA yielded a transgenic heterotetrameric enzyme with similar properties to the purified potato tuber enzyme. It was also found that the extended small subunit expressed by itself exhibited high enzyme activity, with lower affinity for activator 3-phosphoglycerate and higher sensitivity toward inorganic phosphate inhibition. It is proposed that a major function of the large subunit of adenosine 5'-diphosphate-glucose pyrophosphorylases from higher plants is to modulate the regulatory properties of the native heterotetrameric enzyme, and the small subunit's major function is catalysis.

Amino Acid Sequence↗
PubMed1970

On the relationship between ribulose diphosphate carboxylase and protochlorophyllide holochrome of Phaseolus vulgaris leaves.

The relationship between ribulose diphosphate carboxylase (3-phospho-d-glycerate carboxy-lyase [dimerizing], EC 4.1.1.39, formerly known as carboxydismutase) and protochlorophyllide holochrome of etiolated Phaseolus vulgaris leaves has been studied.A procedure for partially selective extraction of the two proteins was devised using tris-HCl buffer first without and then with Triton X-100. Ribulose diphosphate carboxylase was readily extracted from etiolated bean leaves without Triton X-100, and protochlorophyllide holochrome was extracted on the addition of Triton X-100. Optimal extraction conditions for protochlorophyllide holochrome have been found to be different for tissues of different ages.

Carboxy-Lyases↗
PubMed1970

Biosynthesis of cytidine diphosphate diglyceride by enzyme preparations from cauliflower.

The activity of the enzyme responsible for the synthesis of cytidine diphosphate diglyceride has been found in the 20,000g pellet of a cauliflower (Brassica oleracea) inflorescence homogenate. Greater enzyme activity was found in the pellet after treatment with 0.02% Triton X-100, or after freezing and thawing. Activity was greater when fluoride was included in the reaction mixture. The enzyme has a pH optimum of 5.6, an optimal Mn(2+) concentration of 1mm and an optimal Mg(2+) concentration of 30 mm. At the optimal concentration, Mn(2+) was 4 times better than Mg(2+). The utilization of biosynthesized cytidine diphosphate diglyceride can be enhanced by addition of inositol, thereby causing the formation of phosphatidyl inositol.

Journal Article↗
PubMed1970

The Biosynthesis of Sucrose and Nucleoside Diphosphate Glucoses in Phaseolus aureus.

Sucrose-phosphate synthetase is detectable only in intact chloroplast preparations of Phaseolus aureus. In contrast, sucrose synthetase and uridine diphosphate glucose (UDP-glucose) pyrophosphorylase activities are low in extracts of photosynthetic tissues of P. aureus but are high in extracts of nonphotosynthetic tissues. Activities for ADP-, dTDP-, CDP-, and GDP-glucose pyrophosphorylases are generally higher in extracts of photosynthetic tissues of P. aureus than in extracts of nonphotosynthetic tissues. The high levels of sucrose synthetase and of UDP-glucose pyrophosphorylase found in dark-grown hypocotyls begin to decline about 4 hours after exposure to light at a rate of 50% every 3 hours.The data suggest that sucrose-phosphate synthetase and sucrose phosphatase are the enzymes responsible for the biosynthesis of sucrose from photosynthetically fixed CO(2), and that the major function of sucrose synthetase is to catalyze the synthesis of UDP-, ADP-, dTDP-, CDP-, and GDP-glucose from translocated sucrose in nonphotosynthetic tissues; in photosynthetic tissues the pyrophosphorylases may replace sucrose synthetase in catalyzing the synthesis of these nucleoside diphosphate glucoses. We offer the suggestion that sucrose synthetase and UDP-glucose pyrophosphorylase play a major role in the uptake and metabolism of sucrose in nonphotosynthetic tissues. Results are presented from preliminary studies on the conversion in vitro of sucrose to glucose 1-phosphate by the coupled reactions of sucrose synthetase and UDP-glucose pyrophosphorylase with highly purified preparations of these enzymes.

Journal Article↗