Non-enzymatic synthesis of the coenzymes,uridine diphosphate glucose and cytidine diphosphate choline,and other phosphorylated metabolic intermediates

The synthesis of uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P) has been accomplished under simulated prebiotic conditions using urea and cyanamide, two condensing agents considered to have been present on the primitive Earth. The synthesis of UDPG was carried out by reacting G1P and UTP at 70 °C for 24 hours in the presence of the condensing agents in an aqueous medium. CDP-choline was obtained under the same conditions by reacting choline phosphate and CTP. G1P and G6P were synthesized from glucose and inorganic phosphate at 70 °C for 16 hours. Separation and identification of the reaction products have been performed by paper chromatography, thin layer chromatography, enzymatic analysis and ion pair reverse phase high performance liquid chromatography. These results suggest that metabolic intermediates could have been synthesized on the primitive Earth from simple precursors by means of prebiotic condensing agents.     

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.     

Preparation of ADP-Glucose with an Enzyme from Arthrobacter simplex

For the purpose of enzymatic preparation of ADP-glucose (ADPG), bacterial screening was performed to find a strain having a high activity of ADPG pyrophosphorylase which catalyzes the synthesis of ADPG from ATP and glucose-1-phosphate. A cell-free extract of Arthrobacter simplex IFO 12069 showed a strong enzyme activity for the synthesis of ADPG, which was isolated from the reaction solution by ion-exchange column chromatography and identified by paper and thin-layer chromatography. The enzyme activity of the bacterium reached a maximum in the late logarithmic phase under aerobic growth conditions. Some factors affecting the ADPG synthesis, e.g. reaction pH, substrate concentrations, divalent cations, inhibitors and activators, were studied with an ammonium sulfate fraction, 30~50% saturation as the enzyme preparation.     

Cyanamide mediated syntheses under plausible primitive earth conditions

The synthesis of palmitoylglycerols in good yields occurs when a solution of glycerol, ammonium palmitate, cyanamide and imidazole is dried and heated at ambient humidity at temperatures ranging from 60 degrees--100 degrees C for 16 h. Much less product is formed in the absence of either or both cyanamide or imidazole. This work suggests that acylglycerols could have been synthesized on the primitive Earth under plausible prebiotic conditions which were similar but not identical to those which have been shown to condense deoxynucleotides into oligodeoxynucleotides and amino acids into peptides.     

The enzymatic transformation of uridine diphosphate glucose into a galactose derivative

Treatment of uridine diphosphate glucose (UDPG) with an enzyme of S. fragilis was found to produce about 25% of a galactose-containing compound. This compound is precipitated with mercuric ions like UDPG, and its migration in chromatography in acid-ethanol is similar. By alkaline treatment it gives, like UDPG, a doubly esterified hexose monophosphate. It is concluded that the compound is uridine diphosphate galactose, and the bearing of this finding on the mechanism of action of UDPG is discussed.     

Poly(adenosine diphosphate ribose) glycohydrolase in Physarum polycephalum.

Poly(adenosine diphosphate ribose) glycohydrolase, which has thus far only been found in mammalian tissues, was found for the first time in the primitive eukaryotic slime mold Physarum polycephalum. The hydrolytic product of poly(adenosine diphosphate ribose) with this enzyme was identified as adenosine diphosphate ribose by paper and thin-layer chromatography. It is likely that the enzyme caused exoglycosidic hydrolysis. The optimal pH of this enzyme was 6.0, and the K_m value was 4.3 μm, as adenosine diphosphate ribose residues of polymer. Adenosine diphosphate ribose, ADP and ATP at a concentration of 0.1mm strongly inhibited the enzyme activity. 3′,5′-Cyclic AMP was inhibitory at a concentration of 1mm. The molecular weight of this enzyme was estimated to be 57,000.     

Purification and characterization of two fructose diphosphate aldolases from Escherichia coli (Crookes'' strain)

Two fructose diphosphate aldolases (EC 4.1.2.13) were detected in extracts of Escherichia coli (Crookes' strain) grown on pyruvate or lactate. The two enzymes can be resolved by chromatography on DEAE-cellulose at pH7.5, or by gel filtration on Sephadex G-200, and both have been obtained in a pure state. One is a typical bacterial aldolase (class II) in that it is strongly inhibited by metal-chelating agents and is reactivated by bivalent metal ions, e.g. Ca(2+), Zn(2+). It is a dimer with a molecular weight of approx. 70000, and the K(m) value for fructose diphosphate is about 0.85mm. The other aldolase is not dependent on metal ions for its activity, but is inhibited by reduction with NaBH(4) in the presence of substrate. The K(m) value for fructose diphosphate is about 20mum (although the Lineweaver-Burk plot is not linear) and the enzyme is probably a tetramer with molecular weight approx. 140000. It has been crystallized. On the basis of these properties it is tentatively assigned to class I. The appearance of a class I aldolase in bacteria was unexpected, and its synthesis in E. coli is apparently favoured by conditions of gluconeogenesis. Only aldolase of class II was found in E. coli that had been grown on glucose. The significance of these results for the evolution of fructose diphosphate aldolases is briefly discussed.     

Adenosine diphosphate glucose pyrophosphorylase, a rate‐limiting step in starch biosynthesis

Starch represents the major component of virtually all plant‐derived foods consumed by man and animal. Hence, a thorough understanding of the starch biosynthetic pathway is critically important not only in understanding the biosynthesis of a major plant storage product, but also in allowing the genetic manipulation of both starch quality and quantity for human benefit. A major goal in these studies has been the identification of key steps in controlling starch levels. Evidence from a number of independent approaches clearly points to the enzyme adenosine diphosphate glucose pyrophosphorylase (AGPase) as a key regulatory step in starch synthesis. Here we highlight and summarize our understanding of this important enzyme.     

A kinetic study on the chemical cleavage of nucleoside diphosphate sugars

Nucleoside diphosphate sugars serve in essential roles in metabolic processes. They have, therefore, been used in mechanistic studies on glycosylation reactions, and their analogues have been synthesised as enzyme and receptor inhibitors. Despite extensive biochemical research, little is known about their chemical reactions. In the present work the chemical cleavage of two different types of nucleoside diphosphate sugars has been studied. UDP-Glc is phosphorylated at the anomeric carbon, whereas in ADP-Rib C-1 is unsubstituted, allowing hence the equilibrium between cyclic hemiacetal and acyclic carbonyl forms. Due to the structural difference, these substrates react via different pathways under slightly alkaline conditions: while UDP-Glc reacts exclusively by a nucleophilic attack of a glucose hydroxyl group on the diphosphate moiety, ADP-Rib undergoes a complex reaction sequence that involves isomerisation processes of the acyclic ribose sugar and results in a release of ADP.     

Synthesis, characterization and some properties of dideoxynucleoside analogs of cytidine diphosphate diacylglycerol.

Phospholipid conjugates of antiretroviral nucleoside analogs have been proposed to have several advantageous features when compared to the parent drugs (Hostetler, K.Y. et al. (1990) J. Biol. Chem. 265, 6112-6117). Here we report on the synthesis of one such type of lipid conjugates, i.e., nucleosides diphosphate diacylglycerols. The syntheses of 3'-azido-3'-deoxythymidine diphosphate diacylglycerol, 3'-deoxythymidine diphosphate diacylglycerol and 2',3'-dideoxycytidine diphosphate diacylglycerol (with different acyl chains) were performed starting from phosphatidic acid and the antiviral nucleoside. A high-performance liquid chromatography procedure for a single step purification of the compounds is presented. The compounds were characterized biochemically, using rat liver enzymes and chemically by phosphorus, fatty acid, ultraviolet, IR and 1H-NMR analyses. Preliminary data on the behaviour in aqueous solution of some of the compounds are presented.     

Disruption of the structural gene for farnesyl diphosphate synthase in Escherichia coli

The chromosomal ispA gene encoding farnesyl diphosphate synthase of Escherichia coli was disrupted by inserting a neo gene cassette. The null ispA mutants were viable. The growth yield of the mutants was 70% to 80% of that of the wild-type strain under aerobic conditions, and was almost the same as the wild-type under anaerobic conditions. The levels of ubiquinone-8 and menaquinone-8 were both significantly lower (less than 13% and 18% of normal, respectively) in the mutants than in the wild-type. The undecaprenyl phosphate level in the mutants was modestly lower (40% to 70% of normal) than in the wild-type strain. Thus the synthesis of all-E-octaprenyl diphosphate, the precursor of ubiquinone-8 and menaquinone-8, was decreased more severely than that of Z,E-mixed undecaprenyl diphosphate, the precursor of undecaprenyl monophosphates, under the conditions where the synthesis of farnesyl diphosphate was decreased. The condensation of isopentenyl diphosphate with dimethylallyl diphosphate was detected in the cell-free extracts of the mutants, although it was 5% of that in the wild-type strain. A low level of farnesyl diphosphate seems to be synthesized in the mutants by other prenyltransferases such as octaprenyl diphosphate synthase or undecaprenyl diphosphate synthase.     

Sucrose metabolism in Sorghum vulgare at ripening

Enzymes associated with sucrose metabolism in root, stem, leaf and grain of Sorghum vulgare Pers. (cv. JS 263) were studied at the ripening stage. Sucrose phosphate synthetase was dominating in the leaf and sucrose synthetase in the grain. Invertases were more active in leaf, root and stem tissues than in grains. The maximum activities of ADPG pyrophosphorylase and UDPG pyrophosphorylase were found in grains and leaves, respectively. Sucrose synthetase from grains catalyses both synthesis and cleavage of sucrose but the two activities differed in their responses to the effect of temperature, pH and type of buffer. The Km values of the enzyme for UDPG, ADPG, GDPG, TDPG and CDPG were 8.5, 5.3, 16.8 2.2 and 10.7 mM, and for UDP and ADP they were 17.2 and 55.0 mM respectively.     

Biosynthesis of trans,trans,trans-geranylgeranyl diphosphate by the cytosolic fraction from rat tissues.

The cytosolic fractions from rat liver, brain, kidney, spleen and testis demonstrate the capacity to synthesize two products from [3H]isopentenyl diphosphate, i.e., farnesyl diphosphate and geranylgeranyl diphosphate. The highest rate of geranylgeranyl diphosphate synthesis was found in brain, testis and spleen, accounting for up to 30% of the total incorporation of radioactivity under optimal conditions. In all tissues examined the geranylgeranyl diphosphate formed was identified as the trans,trans,trans-isomer. The ratio of geranylgeranyl diphosphate to farnesyl diphosphate produced was specific for the tissue investigated and could be altered by the addition of divalent cations. The results in this study demonstrate the presence of a specific trans,trans,trans-geranylgeranyl diphosphate synthetase showing high affinity for farnesyl diphosphate.     

The metal ion catalyzed decomposition of nucleoside diphosphate sugars.

The metal ion catalysed decomposition of the nucleotide diphosphate sugars, uridine diphosphate glucose, uriding diphosphate galactose, uridine diphosphate N-acetylglucosamine, guanosine diphosphate mannose, and guanosine diphosphate fucose (UDPGlc, UDPGal, UDPGlc-NAc, GDPMan, and GDPFuc, respectively), has been studies as a function of pH. UDPDlc and UDPGal decompose readily to the a,2-cycle phosphate derivative of the sugar and uridine 5'-phosphoric acid (UMP) in the presence of Mn2+. Under all conditions tested, UDPGal decomposes two to three times more rapidly than does UDPGlc. GDPFuc is slowly degraded to free fucose under similar conditions; the other nucleotide diphosphate sugars are stable. The rate of reaction increases with increasing hydroxide ion concentration from pH 6.5 to 7.9 and with metal ion concentration from 10 to 200 mm. Several metal ions are effective catalysts; at pH 7.5 WITH 20 mM UDPGal and 20 mM metal ion, the following apparent first-order rate constants (min-1 x 10(4)) were obtained: Eu3+ 700; Mn2+, 70; Co2+ 27; Zn2+, 22; Ca2+, 3.0; Cu2+, 2.4; and Mg2+, 0. It appears that Mn2+ concentrations that have been used in studies with nucleotide diphosphate sugars at neutral pH can catalyze significant decomposition leading to erroneous interpretation of kinetic and incorporation experiments.     

Enzymatic synthesis of isoprene from dimethylallyl diphosphate in aspen leaf extracts

Aspen (Populus tremuloides Michx.) leaf extracts contain a newly discovered enzyme activity that catalyzes the magnesium ion-dependent elimination of diphosphate from dimethylallyl diphosphate with rearrangement to form isoprene (2-methyl, 1-3-butadiene). This isoprene synthase activity has been partially purified. The nonenzymatic reaction of dimethylallyl diphosphate to isoprene, known to be acid catalyzed, may be insignificant at physiological pH. In contrast, the enzymatic reaction may be responsible for the majority of light-dependent isoprene production by isoprene-emitting plants.     

Enzymatic synthesis of cytidine diphosphate diglyceride

Evidence is presented for the enzymatic formation of cytidine diphosphate diglyceride in microsomal preparations from guinea pig liver according to the reaction: CTP + phosphatidic acid right harpoon over left harpoon CDP-diglyceride + p-O-P. Conditions have been found in which the incorporation of labeled CTP into CDP-diglyceride is almost entirely dependent upon added phosphatidic acid. The incorporation of CMP into lipid is very slight. A substantial net synthesis of CDP-diglyceride takes place under these conditions. Some properties of the enzyme system are described.     

Glycosyl transfer to an acceptor lipid from insects.

Insect extracts were found to contain a lipid which becomes glycosylated when incubated with uridine diphosphate glucose or uridine diphosphate N-acetylglucosamine and microsomal enzymes of rat liver. The behaviour of the lipid on column or thin-layer chromatography and its stability to acid were equal to those of dolichol monophosphate. The glycosylated compounds were acid labile. Treatment with alkali of the acetylglucosaminyl compound produced a substance that migrated like a hexose phosphate on electrophoresis and that liberated acetylglucosamine on treatment with alkaline phosphatase. The behaviour of the insect glucosylated lipid on thin-layer chromatography and its stability to phenol were similar to dolichol monophosphate glucose and different from ficaprenyl monophosphate glucose. It is concluded that the insect glycosyl acceptor lipid is an α saturated polyprenyl phosphate.     

Cyanamide mediated syntheses under plausible primitive earth conditions

When an aqueous solution (pH 7.0) of 3H deoxythymidine 5'-triphosphate, deoxythymidine 5'-phosphate, 4-amino-5-imidazolecarboxamide, cyanamide and ammonium chloride was dried and heated at 60 degrees C for 18 h, oligomers were obtained in a yield of approximately 80%. After the chemical degradation of any pyrophosphate bonds present in these oligomers, linear polynucleotides of up to 7-8 units in length were isolated by DEAE cellulose column chromatography and identified by enzymatic digestion procedures. The di- and trinucleotide fractions were degraded 87% and 100% by snake venom phosphodiesterase and 39% and 9% by spleen phosphodiesterase. This synthesis of deoxythymidine oligonucleotides was conducted under potentially prebiotic conditions and may offer a possible method for the synthesis of deoxyoligonucleotides on the primitive Earth.     

Efficient biosynthesis of uridine diphosphate glucose from maltodextrin by multiple enzymes immobilized on magnetic nanoparticles

Uridine diphosphate glucose (UDP-Glc) serves as a glucosyl donor in many enzymatic glycosylation processes. This paper describes a multiple enzyme, one-pot, biocatalytic system for the synthesis of UDP-Glc from low cost raw materials: maltodextrin and uridine triphosphate. Three enzymes needed for the synthesis of UDP-Glc (maltodextrin phosphorylase, glucose-1-phosphate thymidylytransferase, and pyrophosphatase) were expressed in Escherichia coli and then immobilized individually on amino-functionalized magnetic nanoparticles. The conditions for biocatalysis were optimized and the immobilized multiple-enzyme biocatalyst could be easily recovered and reused up to five times in repeated syntheses of UDP-Glc. After a simple purification, approximately 630 mg of crystallized UDP-Glc was obtained from 1 l of reaction mixture, for a moderate yield of around 50% (UTP conversion) at very low cost.     

Characterization of adenosine diphosphate glucose pyrophosphorylases from developing maize seeds

Electrophoretic examination of 22-day-old, normal maize (Zea mays L.) endosperm extracts revealed two zones of adenosine diphosphate glucose pyrophosphorylase activity. The enzymes are identical in terms of Km for glucose 1-phosphate and the effect of 3-phosphoglyceric acid on apparent Km for glucose 1-phosphate. Both enzymatic activities increase with increasing doses of the functional alleles at the shrunken-2 and brittle-2 loci. Molecular weight differences between the two electrophoretic species were inferred from sucrose gradient centrifugation. It is suggested that the two bands of activity represent different aggregation states of the same enzyme because under different extraction conditions, only one enzyme is found. Molecular weight estimates of 237,000 and 253,000 were obtained for the smaller enzyme. It is suggested that this enzyme is an aggregate of several subunits. Comparison of the embryo and endosperm pyrophosphorylases showed the embryo activity to be more heat stable and probably independent of direct shrunken-2 or brittle-2 control.