Izumoring: a strategy for bioproduction of all hexoses

Izumoring is a new concept for the bioproduction of all hexose sugars - 16 aldohexoses, eight ketohexoses and 10 hexitols -- using enzymatic and microbiological reactions. The biocatalysts necessary for the strategy were (i) d-tagatose 3-epimerase [which epimerizes all ketohexoses at C-3 of the sugar], (ii) polyol dehydrogenases [which catalyze oxidation-reduction reactions between ketohexoses and the corresponding hexitols], and (iii) aldose isomerases [which catalyze isomerization reactions between aldohexoses and corresponding aldoketoses]. All ketohexoses, aldohexoses and hexitols may be arranged in a symmetric ring connected by the biochemical reactions, allowing the design for the bioproduction of all 34 hexose sugars. The ring shows there are four entrances to the l-hexose world from the natural d-hexoses. The Izumoring ring shows clearly the relationship and the position of all 34 six carbon sugars.     

Prebiotic sugar synthesis: Hexose and hydroxy acid synthesis from glyceraldehyde catalyzed by iron(III) hydroxide oxide

Summary Iron(III) hydroxide oxide [Fe(OH)O] efficiently catalyzed the condensation of 25 MM dl-glyceraldehyde to ketohexoses at 25°C (pH 5–6). At 16 days the yields were sorbose (15.2%), fructose (12.9%), psicose (6.1%), tagatose (5.6%), and dendroketose (2.5%) with 19.6% of triose unreacted. Analysis at 96 days showed no decomposition of hexoses. Under these conditions Fe(OH)O also catalyzed the isomerization and rearrangement of glyceraldehyde to dihydroxyacetone and lactic acid, respectively. In these reactions, about 10% of the glyceraldehyde was oxidized to glyceric acid with concurrent reduction of the iron(III) to iron(II). The partial reduction of Fe(OH)O did not noticeably reduce its ability to catalyze hexose synthesis. The relationship of these results to prebiotic sugar synthesis is discussed.     

Catalysis of the dephosphorylation of an ATP-molybdate complex by calcium and magnesium ions

Calcium and magnesium catalyze the dephosphorylation of a molybdate complex of adenosine triphosphate but not the corresponding molybdate complex of adenosine disphosphate. We conclude that catalysis of breaking the bond between the beta and gamma phosphates involves metal chelation of the alpha and beta phosphates. ATP hydrolysis with calcium was stimulated by phosphate, apparently because of formation of a calcium-phosphomolybdate complex. The reaction with magnesium, which does not form a comparable complex, was not affected by phosphate. Strontium, cadmium, and barium behaved like calcium. The reactions with transition metal cations showed autoinhibition.     

Hexose phosphates as regulators of hepatic glycogen synthase phosphatases

The activity of glycogen synthase phosphatase from smooth endoplasmic reticulum of liver was stimulated markedly by galactose-6- and fructose-6-phosphates and to a lesser extent by glucose-1- and 2-deoxyglucose-6-phosphates. The synthase phosphatase of liver cytosol showed strong activation by glucose-1-, glucose-6- and fructose-6-phosphates and smaller activation by galactose-6- and 2-deoxyglucose-6-phosphates. Kinetic analysis showed that the activators did not affect the Km for glycogen synthase D, for either enzyme. The mechanism of activation of the two phosphatases by hexose phosphates appears to be by combination of the activator at a specific activator site on the enzyme rather than by substrate modulation. It is concluded that certain hexose phosphates, particularly fructose-6-phosphate and glucose-1-phosphate, can function as regulators of hepatic synthase phosphatase activity, and that this may explain the ability of elevated blood glucose to increase both glycogen synthase I activity and glycogen synthesis in the liver.     

Catalysis of proline isomerization during protein-folding reactions

The enzyme peptidylprolyl cis-trans isomerase (PPI) is known to catalyze proline isomerization in short proline-containing peptides. If PPI can be shown to generally catalyze isomerization of proline residues in proteins, then it would be a valuable diagnostic reagent for recognition of isomerization, which has proven to be extremely difficult to characterize by other methods. In this study, the catalytic effect of PPI on the slow refolding reactions of seven different proteins has been studied, and in only two cases (RNase T1 and cytochrome c) could significant catalysis be seen. PPI also caused no enhancement in the rate for the 'subtle' conformational changes of native concanavalin A or native Fragment I of prothrombin, which have been suggested to be rate-limited by proline isomerization. There was a small effect of PPI observed for the generation of native RNAase A from the fully-reduced form when the glutathione concentration was low. The conclusion from these studies is that PPI can weakly catalyze some protein processes which are rate-limited by proline isomerization, but probably exhibits no measureable catalysis toward others. This somewhat limits the usefulness of PPI as a diagnostic reagent for proline isomerization.     

Properties of two phosphatases and a cyclic phosphodiesterase of Salmonella typhimurium.

The properties of three phosphatases from Salmonella typhimurium have been examined. A cyclic 2',3'-nucleotide phosphodiesterase (EC 3.1.4.d) hydrolyzes cyclic 2',3'-purine and -pyrimidine nucleotides, as well as 3'-mononucleotides, and has a pH optimum of about 7.5. It requires divalent cations for activity and has a molecular weight of 67,000. Acid hexose phosphatase (EC 3.1.2.2) possesses activity towards hexose phosphates as well as other sugar phosphates. The enzyme is apparently a dimer of 37,000-dalton subunits. Nonspecific acid phosphatase (EC 3.1.3.2) hydrolyzes a variety of phosphate esters, including nucleotides and sugar phosphates. The enzyme also hydrolyzes the phosphoric anhydride bonds of pyrophosphate and nucleotides. Michaelis constants of the nonspecific acid phosphatase for several of its substrates are in the 1 to 2 mM range. Nonspecific acid phosphatase is a dimer of 27,000-dalton subunits.     

The control of pyruvate kinases of Escherichia coli. II. Effectors and regulatory properties of the enzyme activated by ribose 5-phosphate.

The pyruvate kinases of Escherichia coli activated by ribose 5-phosphate (RP) has been partially purified. The active form of the enzyme has a molecular weight of about 180 000 as judged by sucrose density gradient centrifugations and Sephadex G-150 chromatography. On dissociation in the absence of sulfhydryl reagents such as dithiothreitol, the enzyme is inactivated and it has a molecular weight of about 110 000. Various substrates and effectors of the enzyme, with the exception of phosphate, do not influence the association-dissociation equilibrium of the enzyme. The enzyme, unlike pyruvate kinases from many other sources, is not activated by potassium ions. Sulfate and phosphate ions are inhibitory to the enzyme. Phosphate seems to be an allosteric inhibitor and its effect is completely antagonized by activators. The enzyme is activated in an allosteric manner by two classes of compounds, nucleoside monophosphates and sugar phosphates of the hexose monophosphate pathway. Amongst the nucleotides, guanosine 5'-phosphate and adenosine 5'-phosphate are the most effective activators. Amongst the hexose monophosphate pathway intermediates, RP is the most powerful activator, with apparent activation constants as low as 1 Mu. Sugar phosphates esterified at C-1 or both terminal positions are entirely ineffective in activation. The effectors act by changing the Michaelis constant for the substrates. Both of the substrates of the enzyme, adenosine diphosphate and phosphoenolpyruvate, yield cooperative-concentration plots in the presence of unsaturating concentrations of the fixed changing substrate. The initial velocity plots for both substrates become hyperbolic in the presence of saturating concentrations of RP.     

Three metal ions participate in the reaction catalyzed by T5 flap endonuclease

Protein nucleases and RNA enzymes depend on divalent metal ions to catalyze the rapid hydrolysis of phosphate diester linkages of nucleic acids during DNA replication, DNA repair, RNA processing, and RNA degradation. These enzymes are widely proposed to catalyze phosphate diester hydrolysis using a "two-metal-ion mechanism." Yet, analyses of flap endonuclease (FEN) family members, which occur in all domains of life and act in DNA replication and repair, exemplify controversies regarding the classical two-metal-ion mechanism for phosphate diester hydrolysis. Whereas substrate-free structures of FENs identify two active site metal ions, their typical separation of > 4 A appears incompatible with this mechanism. To clarify the roles played by FEN metal ions, we report here a detailed evaluation of the magnesium ion response of T5FEN. Kinetic investigations reveal that overall the T5FEN-catalyzed reaction requires at least three magnesium ions, implying that an additional metal ion is bound. The presence of at least two ions bound with differing affinity is required to catalyze phosphate diester hydrolysis. Analysis of the inhibition of reactions by calcium ions is consistent with a requirement for two viable cofactors (Mg2+ or Mn2+). The apparent substrate association constant is maximized by binding two magnesium ions. This may reflect a metal-dependent unpairing of duplex substrate required to position the scissile phosphate in contact with metal ion(s). The combined results suggest that T5FEN primarily uses a two-metal-ion mechanism for chemical catalysis, but that its overall metallobiochemistry is more complex and requires three ions.     

Glutathione transferases. Catalysis of nucleophilic reactions of glutathione

Homogeneous preparations of the glutathione transferases from rat liver have been tested for their ability to catalyze a number of diverse nucleophilic reactions of GSH. Although disulfide interchange with GSSG or L-cystine, and cis-trans isomerization of maleic acid, are clearly promoted by thiols in solution, the reactions were not catalyzed by the glutathione transferases. In contrast, certain more hydrophobic analogs of these compounds were found to serve as substrates. The transferases also catalyze the glutathione-dependent release of p-nitrophenol from p-nitrophenyl acetate and p-nitrophenyl trimethylacetate. These observations are consistent with the formulation that catalysis may result from close juxtaposition of sufficiently electrophilic, nonpolar compounds with GSH on the enzyme surface.     

Aminoglycoside-RNA interactions

The structural and physico-chemical parameters promoting the binding of aminoglycosides to RNAs are becoming clear. The strength of the interaction is dominated by electrostatics, with the positively charged aminoglycosides displacing metal ions. Although aminoglycosides inhibit most known ribozymes, aminoglycosides or polyamines are able to catalyze specific RNA cleavage in the absence of metal ions.     

A comparative study of metabolite levels in plant leaf material in the dark

Metabolite levels have been compared in the dark and during photosynthesis in leaves and protoplasts from spinach, pea, wheat and barley. In protoplasts the subcellular distribution was also studied. The levels of triose phosphates and sugar bisphosphates were high in the light and low in the dark. The hexose phosphates and 3-phosphoglycerate levels in the dark were very variable depending on the plant material. In most conditions, hexose phosphates and triose phosphates were mainly in the extrachloroplast compartment, while 3-phosphoglycerate and the sugar bisphosphates were mainly in the chloroplast compartment. Leaves always had a very low triose phosphate: 3-phosphoglycerate ratio in the dark, but in protoplasts this ratio was higher. Detailed studies with spinach showed that metabolite levels were very dependent on the availability of carbohydrate in the leaf, particularly starch. Starch mobilisation is not controlled just by the availability of inorganic phosphate and accumulation of phosphorylated intermediates. Hydrolysis of starch may provide precursors for sucrose synthesis while phosphorolysis leads to provision of substrates for respiration. Starch breakdown generates high enough levels of hexose phosphate to support substantial rates of sucrose synthesis in the dark. Respiration is not greatly increased when metabolite levels are high during starch mobilisation. Higher levels of metabolites shorten the length of the induction phase of photosynthesis.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - Fru2,6bisP fructose-2,6-bisphosphate - NMR nuclear magnetic resonance - PGA 3-phosphoglyceric acid - Pi inorganic phosphate - RuBP ribulose-1,5-bisphosphate - UDPGlc uridine-5-diphosphate glucose     

Measurement of Metabolites Associated with Nonaqueously Isolated Starch Granules from Immature Zea mays L. Endosperm

Starch granules with associated metabolites were isolated from immature Zea mays L. endosperm by a nonaqueous procedure using glycerol and 3-chloro-1,2-propanediol. The soluble extract of the granule preparation contained varying amounts of neutral sugars, inorganic phosphate, hexose and triose phosphates, organic acids, adenosine and uridine nucleotides, sugar nucleotides, and amino acids. Based on the metabolites present and on information about translocators in chloroplast membranes, which function in transferring metabolites from the chloroplast stroma into the cytoplasm, it is suggested that sucrose is degraded in the cytoplasm, via glycolysis, to triose phosphates which cross the amyloplast membrane by means of a phosphate translocator. It is further postulated that hexose phosphates and sugars are produced from the triose phosphates in the amyloplast stroma by gluconeogenesis with starch being formed from glucose 1-phosphate via pyrophosphorylase and starch synthase enzymes. The glucose 1-phosphate to inorganic phosphate ratio in the granule preparation was such that starch synthesis by phosphorylase is highly unlikely in maize endosperm.     

Micromolar HgCl2 concentrations transitorily duplicate the ATP level in Saccharomyces cerevisiae cells

Low concentrations of HgCl2 elicited, in Saccharomyces cerevisiae, a transitory increase in the ATP level followed by a decrease of its concentration, until almost disappearance. At 1 microM HgCl2, the increase in ATP lasted for about 30 min, while at 10 microM the increase was only observed in the first 5 min of treatment. The initial burst of ATP was accompanied by a decrease in the level of hexose phosphates, whereas during the decrease of ATP an increase in the inosine and hexose phosphates levels took place. The treatment with HgCl2 inhibited the plasma membrane proton ATPase but not the activities of hexokinase or 6-phosphofructokinase.     

Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells

The aim of this work was to establish the influence of fructose 2,6-bisphosphate (Fru-2,6-P₂) on non-photosynthetic carbohydrate metabolism in plants. Heterotrophic callus lines exhibiting elevated levels of Fru-2,6-P₂ were generated from transgenic tobacco (Nicotiana tabacum L.) plants expressing a modified rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Lines containing increased amounts of Fru-2,6-P₂ had lower levels of hexose phosphates and higher levels of 3-phosphoglycerate than the untransformed control cultures. There was also a greater redistribution of label into the C6 position of sucrose and fructose, following incubation with [1-¹³C]glucose, in the lines possessing the highest amounts of Fru-2,6-P₂, indicating a greater re-synthesis of hexose phosphates from triose phosphates in these lines. Despite these changes, there were no marked differences between lines in the metabolism of ¹⁴C-substrates, the rate of oxygen uptake, carbohydrate accumulation or nucleotide pool sizes. These data provide direct evidence that physiologically relevant changes in the level of Fru-2,6-P₂ can affect pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in vivo, and are consistent with PFP operating in a net glycolytic direction in the heterotrophic culture. However, the results also show that activating PFP has little direct effect on heterotrophic carbohydrate metabolism beyond increasing the rate of cycling between hexose phosphates and triose phosphates. Received: 29 March 2000 / Accepted: 13 June 2000     

Stability and isomerization of complexes formed by metal ions and cytosine isomers in aqueous phase

We present a systematic study of the stability of the formation of complexes produced by four metal ions (M^+/2+) and 14 cytosine isomers (C_n). This work predicts theoretically that predominant product complexes are associated with higher-energy C₄M^+/2+ and C₅M^+/2+ rather than the most stable C₁M^+/2+. The prediction resolves successfully several experimental facts puzzling two research groups. Meanwhile, in-depth studies further reveal that direct isomerization of C₁?C₄ is almost impossible, and also that the isomerization induced by either metalation or hydration, or by a combination of the two unfavorable. It is the single water molecule locating between the H1(?N1) and O2 of the cytosine that plays the dual roles of being a bridge and an activator that consequently improves the isomerization greatly. Moreover, the cooperation of divalent metal ion and such a monohydration actually leads to an energy-free C₁←C₄ isomerization in the gas phase. Henceforth, we are able to propose schemes inhibiting the free C₁←C₄ isomerization, based purely on extended hydration at the divalent metal ion.

Characterization of a regulatory mutant of fructose 1,6-bisphosphatase in Saccharomyces carlsbergensis.

Summary The fdp mutation has been localized on the genome of Saccharomyces carlsbergensis, on chromosome II, between lys2 and tyr1, at a map distance of 31 centimorgan from lys2.Since the fdp mutant does not grow on glucose, fructose, mannose and sucrose, hexose transport and a number of enzymes of carbon metabolism were tested, but no significant differences could be found between the wild type and the mutant. Only the regulatory properties of glycogen synthetase are changed in the mutant, but it is doubtfull whether this can explain its phenotype.The disorganization of carbon metabolism of the mutant upon addition of glucose to the medium was analyzed in more detail. The most prominent feature observed until now is the accumulation of free glucose and hexose phosphates in the cell. This result indicates that somehow the feedback control between hexose transport and metabolism is impaired. Hexose phosphates are known to be toxic to many cells, including yeast. Therefore, accumulation of hexose phosphates in the presence of glucose in the medium, can explain the absence of growth on this carbon source.     

Evidence that aldolase and D-arabinose 5-phosphate are components of pentose pathway reactions in liver in vitro

An immunochemical procedure involving the reaction of liver aldolase antibody and rat liver enzyme preparation shows that conversion of ribose 5-P to hexose 6-P by reactions of the non-oxidative pentose pathway fails to occur in the absence of aldolase activity. Radioautography of pentose pathway products formed by liver enzyme catalysis of [U-14C] arabinose 5-P and unlabelled ribose 5-P illustrates the incorporation of 14C into ketopentose, sedoheptulose, fructose and glucose phosphates. There is approximate congruity of the mole specific radioactivity of the pentose and hexose phosphates. These findings are consistent with the proposal that L-pentose pathway reactions constitute the non-oxidative segment of the pathway in liver.     

The plastidic pentose phosphate translocator represents a link between the cytosolic and the plastidic pentose phosphate pathways in plants

Plastids are the site of the reductive and the oxidative pentose phosphate pathways, which both generate pentose phosphates as intermediates. A plastidic transporter from Arabidopsis has been identified that is able to transport, in exchange with inorganic phosphate or triose phosphates, xylulose 5-phosphate (Xul-5-P) and, to a lesser extent, also ribulose 5-phosphate, but does not accept ribose 5-phosphate or hexose phosphates as substrates. Under physiological conditions, Xul-5-P would be the preferred substrate. Therefore, the translocator was named Xul-5-P/phosphate translocator (XPT). The XPT shares only approximately 35% to 40% sequence identity with members of both the triose phosphate translocator and the phosphoenolpyruvate/phosphate translocator classes, but a higher identity of approximately 50% to glucose 6-phosphate/phosphate translocators. Therefore, it represents a fourth group of plastidic phosphate translocators. Database analysis revealed that plant cells contain, in addition to enzymes of the oxidative branch of the oxidative pentose phosphate pathway, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase in both the cytosol and the plastids, whereas the transketolase and transaldolase converting the produced pentose phosphates to triose phosphates and hexose phosphates are probably solely confined to plastids. It is assumed that the XPT function is to provide the plastidic pentose phosphate pathways with cytosolic carbon skeletons in the form of Xul-5-P, especially under conditions of a high demand for intermediates of the cycles.     

Changes in the concentrations of some phosphorylated intermediates and stimulation of glycolysis in liver slices

1. The concentrations of some phosphorylated glycolytic intermediates and of NADH were measured in glycolysing rat liver slices. 2. In anaerobically incubated liver slices the concentration of hexose monophosphates decreases during the first 20min. of incubation, whereas the concentrations of fructose diphosphate and triose phosphates increase progressively. 3. In liver slices from fed rats, previously exposed to oxygen, the stimulated anaerobic glycolysis is accompanied by an increase in the concentration of hexose monophosphates; fructose diphosphate and triose phosphates maintain the concentrations reached at the end of the aerobic preincubation. 4. The same pattern in the concentration of glycolytic phosphorylated intermediates is seen under all conditions where aerobic preincubation brings about a stimulation of anaerobic glycolysis. A similar pattern is also found in liver slices from fed rats incubated anaerobically in the presence of fructose; these slices display a high glycolytic activity, which is not further affected by previous aerobic incubation. 5. The concentration of NADH decreases in liver slices during exposure to oxygen; during the subsequent anaerobic glycolysis the concentration increases but is always lower in preincubated than in non-preincubated liver slices. 6. The results of the present experiments suggest that the limiting step mainly affected by the preliminary exposure to oxygen might be at the level of the utilization of triose phosphates.     

Thiophilic metal ion rescue of phosphorothioate interference within the Tetrahymena ribozyme P4-P6 domain

Divalent metal ions are essential for the folding and catalytic activities of many RNAs. A commonly employed biochemical technique to identify metal-binding sites in RNA is the rescue of Rp alpha-phosphorothioate (PS) interference by the addition of soft divalent metal ions. To access the ability of such experiments to accurately identify metal-ion coordinations within a complex RNA fold, we report metal-rescue results from the Tetrahymena group I intron P4-P6 domain, where the location and coordination of five divalent metal ions have been determined by X-ray crystallography [J.H. Cate et al., Nat Struct Biol, 1997, 4:553]. We used a native gel mobility-shift to assay for P4-P6 folding in the presence of various divalent metal ions, and found that even moderate concentrations of Mn2+ (> or =0.5 mM) can rescue PS interference at sites that do not coordinate metal ions within the P4-P6 crystal structure. To control for such effects, 2'-deoxynucleotide interference was used to titrate the Mn2+ concentration to a level that produces metal-ion-specific rescue (0.3 mM). This concentration of Mn2+ specifically rescued four of the six metal-dependent phosphorothioate effects within the RNA domain, including PS interference resulting from outer-sphere coordination to the metals. Both sites that were not specifically rescued make inner-sphere metal-ion coordinations. Cd2+ and Zn2+ afforded rescue at a smaller subset of the six metal-specific PS sites, though again phosphates making outer-sphere coordinations to metal ions were rescued preferentially. These data on P4-P6 domain folding reinforce the need for caution when interpreting metal-rescue experiments.