The guanosine 5'-diphosphate D-mannose: guanosine 5'-diphosphate L-galactose epimerase of Chlorella pyrenoidosa. Chemical synthesis of guanosine 5'-diphosphate L-galactose and further studies of the enzyme and the reaction it catalyzes.

An enzyme from extracts of the green alga Chlorella pyrenoidosa that catalyzes the reversible epimerization of guanosine 5′-diphosphate d-mannose to guanosine 5′-diphosphate l-galactose was further purified. The substrate guanosine 5′-diphosphate l-galactose was made chemically by the morpholidate procedure. An improved method was developed for the synthesis of an intermediate in that process, β-l-galactopyranosyl phosphate, via an orthoester of l-galactose. Various characteristics of the enzyme and the reaction it catalyzes were studied. A new method using gas-liquid chromatography was introduced for following the course of the reaction with unlabeled substrates.     

The Synthesis of Guanosine 5'-Diphosphate l-Fucose from Guanosine 5'-Diphosphate 3,5-d-[H]Mannose Catalyzed by an Enzyme Extract from Fruits of the Flax

An enzyme system from fruits of the flax plant is described that catalyzes the synthesis of the sugar nucleotide guanosine 5'-diphosphate l-fucose from guanosine 5'-diphosphate d-mannose with the intermediate formation of guanosine 5'-diphosphate 4-keto-6-deoxy-d-mannose. Tritium from-[(3)H]H(2)O was incorporated into the l-fucose portion of the sugar nucleotide in the course of the reaction, and tritium at the 3,5-carbons of the d-mannose moiety of GDP-d-mannose was exchanged with protons in the medium. These results support a mechanism of synthesis analogous to that proposed for the formation of l-rhamnose and other 6-deoxy sugars.     

Interaction of guanosine 5′-diphosphate, 2′-(or 3′-) diphosphate(ppGpp) with elongation factors from

The interactions of guanosine 5′-diphosphate, 2′-(or 3′-) diphosphate(ppGpp) with the polypeptide elongation factors Tu(EF Tu) and G(EF G) have been studied. The data indicate that ppGpp binds with EF Tu to form an EF Tu-ppGpp complex, and inhibits, in a competitive manner, the exchange reaction of Tu-GDP and ³H-GDP. The ribosome-dependent GTPase reaction catalyzed by EF G is also depressed by ppGpp.     

Recognition of RNA cap in the Wesselsbron virus NS5 methyltransferase domain: implications for RNA-capping mechanisms in Flavivirus

The mRNA-capping process starts with the conversion of a 5′-triphosphate end into a 5′-diphosphate by an RNA triphosphatase, followed by the addition of a guanosine monophosphate unit in a 5′-5′ phosphodiester bond by a guanylyltransferase. Methyltransferases are involved in the third step of the process, transferring a methyl group from S-adenosyl-l-methionine to N7-guanine (cap 0) and to the ribose 2′OH group (cap 1) of the first RNA nucleotide; capping is essential for mRNA stability and proper replication. In the genus Flavivirus, N7-methyltransferase and 2′O-methyltransferase activities have been recently associated with the N-terminal domain of the viral NS5 protein. In order to further characterize the series of enzymatic reactions that support capping, we analyzed the crystal structures of Wesselsbron virus methyltransferase in complex with the S-adenosyl-l-methionine cofactor, S-adenosyl-l-homocysteine (the product of the methylation reaction), Sinefungin (a molecular analogue of the enzyme cofactor), and three different cap analogues (GpppG, ^N7MeGpppG, and ^N7MeGpppA). The structural results, together with those on other flaviviral methyltransferases, show that the capped RNA analogues all bind to an RNA high-affinity binding site. However, lack of specific interactions between the enzyme and the first nucleotide of the RNA chain suggests the requirement of a minimal number of nucleotides following the cap to strengthen protein/RNA interaction. Our data also show that, following incubation with guanosine triphosphate, Wesselsbron virus methyltransferase displays a guanosine monophosphate molecule covalently bound to residue Lys28, hinting at possible implications for the transfer of a guanine group to ppRNA. The structures of the Wesselsbron virus methyltransferase complexes obtained are discussed in the context of a model for N7-methyltransferase and 2′O-methyltransferase activities.     

A bulky platinum triamine complex that reacts faster with guanosine 5′-monophosphate than with N-acetylmethionine

A bulky platinum triamine complex, [Pt(Me₅dien)(NO₃)]NO₃ (Me₅dien = N,N,N′,N′,N′′-pentamethyldiethylenetriamine) has been prepared and reacted in D₂O with N-acetylmethionine (N-AcMet) and guanosine 5′-monophosphate (5′-GMP); the reactions have been studied using ¹H NMR spectroscopy. Reaction with 5′-GMP leads to two rotamers of [Pt(Me₅dien)(5′-GMP-N7)]⁺. Reaction with N-AcMet leads to formation of [Pt(Me₅dien)(N-AcMet-S)]⁺. When a sample with equimolar mixtures of [Pt(Me₅dien)(D₂O)]²⁺, 5′-GMP, and N-AcMet was prepared, [Pt(Me₅dien)(5′-GMP-N7)]⁺ was the dominant product observed throughout the reaction. This selectivity is the opposite of that observed for a similar reaction of [Pt(dien)(D₂O)]²⁺ with 5′-GMP and N-AcMet. To our knowledge, this is the first report of a platinum(II) triamine complex that reacts substantially faster with 5′-GMP than with N-AcMet; the effect is most likely due to steric clashes between the methyl groups of the Me₅dien ligand and the N-AcMet.     

Metal ion coordination to 2′ functionality of guanosine mediates substrate-guanosine coupling in group I ribozymes: implications for conserved role of metal ions and for variability in RNA folding in ribozyme catalysis

Divalent metal ions are necessary in the self splicing reaction of group I introns, and we report that metal interaction to the 2′ position of guanosine for the Azoarcus ribozyme is required for catalysis. Moreover, this metal coordination promotes the guanosine-substrate coupled binding to the ribozyme, which is another conserved feature seen across phylogenetic boundaries. Typically there is a 4-9-fold difference in binding of G to E_free versus E · S. In the Tetrahymena ribozyme’s case this substrate-guanosine communication was attributed to conformational change(s) that lead to cooperative binding of the two cofactors which is almost nonexistent at low temperatures (4 °C). In the prokaryotic Azoarcus ribozyme we also see a 4-5-fold difference in binding of the guanosine/substrate to E_free versus E · G or E · S at 10 °C that is attributed to guanosine-substrate coupling. This coupling is diminished when the metal (Mg²⁺) coordination to the 2′ is disrupted with use of 2′-amino-2′-deoxyguanosine. The coupling is restored when softer Mn²⁺ ions are added to the buffer. This evidence generalizes a model for group I ribozyme catalysis that involves metal coordination to the 2′ position of guanosine. However, we see one striking difference in that the guanosine-substrate coupling is reversed. In the Azoarcus system (10 °C) the guanosine/substrate binds 5-fold more tightly to E_free than to E · S or E · G, which is the opposite for Tetrahymena even when the later is run at 4 °C. One implication for this difference in coupling is that the Azoarcus is in a folded state well accommodated for guanosine or substrate binding. This initial binding actually causes a conformational change that retards the subsequent binding of the second cofactor, which contrasts what was found for the Tetrahymena ribozyme. These results indicate that while the role for the metal ions in the chemical catalysis is conserved across phylogenetic boundaries, there is variability in the folding pattern of the ribozyme that leads to phosphoryl transfer.     

Relative levels of guanosine 5′-diphosphate 3′-diphosphate (ppGpp) in some N2 fixing bacteria during derepression and repression of nitrogenase

Addition of ammonium to N₂ fixing cultures of Azotobacter vinelandii, Klebsiella pneumoniae and Clostridium pasteurianum rapidly reduced the intracellular levels of guanosine 5-diphosphate 3-diphosphate (ppGpp) by 70–90%. This change might reflect a regulatory role of ppGpp in nitrogen metabolism.Abbreviations ppGpp guanosine 5-diphosphate 3-diphosphate     

Polysaccharide components of the seed-coat mucilage from hyptis suaveolens

The mucilage isolated from the seed coat of Hyptis suaveolens contains l-fucose, d-xylose, d-mannose, d-galactose, d-glucose and 4-O-methyl-d-glucuronic acid in the mol ratios 1.0:2.5:1.5:7.0:12.5:1.1. Fractionation of the mucilage with Fehling's solution gave a neutral and an acidic polysaccharide. The neutral polysaccharide appears to be homogeneous and is composed of d-mannose, d-galactose and d-glucose in the mol ratios 1.0:4.5:7.5. The acidic polysaccharide is composed of l-fucose, d-xylose and 4-O-methyl-d-glucuronic acid in the mol ratios 1.0:2.5:1.1. It is homogeneous on gel filtration, DEAE-cellulose chromatography, sedimentation analysis and electrophoresis.     

Very slow growth of Bacillus polymyxa: Stringent response and maintenance energy

Bacillus polymyxa grown in a recycling fermentor shows the same behavior previously observed with Escherichia coli: 3 successive growth phases. In the last 2 phases the growth rate is linear and the apparent maintenance energy demand rate and the molar growth yield are both independent of the specific growth rate, , and of the cells mass. The final phase of very slow growth is an indefinitely prolonged state of strong, stringent control, the regulatory system based on guanosine 3-diphosphate 5-diphosphate, and guanosine 3-diphosphate 5-triphosphate. The maximum cost of this stringent response is calculated to be 9% of the energy available to these energy-limited cells. There is a further energy cost contained in substantial amounts of DNA, RNA, and protein released from the cells during the latter 2 growth phases. The cost of production of these extra cellular anabolites ranges from 8–11% of the available energy.After a carbon-energy upshift in phase 3, the population growth rate immediately returned to that of early phase 2 growth, 50 h or more earlier.If maintenance energy is considered as energy expended by cells to maintain homeostasis, catabolic capacity, or anabolic potential, then the cost of stringent control — which preserves the fidelity of protein synthesis in slowly growing cells — must be considered a maintenance energy cost.Abbreviations GPR glucose provision rate - F_R medium flow rate - S_R substrate concentration - V_F fermentor volume - F_S filtrate removal rate - ppGpp guanosine 3-diphosphate 5-diphosphate - pppGpp guanosine 3-diphosphate 5-triphosphate     

Binding of palladium(II) complexes to guanine, guanosine or guanosine 5-monophosphate in aqueous solution: potentiometric and NMR studies

The interaction of guanine, guanosine or 5^′-GMP (guanosine 5^′-monophosphate) with [Pd(en)(H₂O)₂](NO₃)₂ and [Pd(dapol)(H₂O)₂](NO₃)₂, where en is ethylenediamine and dapol is 2-hydroxy-1,3-propanediamine, were studied by UV-Vis, pH titration and ¹H NMR. The pH titration data show that both N1 and N7 can coordinate to [Pd(en)(H₂O)₂]²⁺ or [Pd(dapol)(H₂O)₂]²⁺. The pK_a of N1-H decreased to 3.7 upon coordination in guanosine and 5^′-GMP complexes, which is significantly lower than that of ∼9.3 in the free ligand. In strongly acidic solution where N1-H is still protonated, only N7 coordinates to the metal ion, but as the pH increases to pH ∼3, ¹H NMR shows that both N7-only and N1-only coordinated species exist. At pH 4-5, both N1-only and N1,N7-bridged coordination to Pd(II) complexes are found for guanosine and 5^′-GMP. The latter form cyclic tetrameric complexes, [Pd(diamine)(μ-N1,N7-Guo]₄⁴⁺ and [Pd(diamine)(μ-N1,N7-5^′-GMP)]₄H_x^(4−x)−, (x=2,1, or 0) with either [Pd(en)(H₂O)₂](NO₃)₂ or [Pd(dapol)(H₂O)₂](NO₃)₂. The pH titration data and ¹H NMR data agree well with the exception that the species distribution diagrams show the initial formation of the N1-only and N1,N7-bridged complexes to occur at somewhat higher pH than do the NMR data. This is due to a concentration difference in the two sets of data.     

Reaction behavior of molybdocene dichloride towards ribonucleosides and ribonucleoside monophosphates: Rare example of sugar coordination

The coordination behavior of Cp₂Mo²⁺ towards the ribonucleosides and ribonucleoside monophosphates uridine, adenosine, cytidine, guanosine, 5′-UMP, 5′-AMP, 5′-CMP and 5′-GMP has been studied in solution in the range 4 ? pD ? 9 using NMR spectroscopy. The ribonucleosides were found to bind Cp₂Mo²⁺ exclusively through the ribose moiety giving rise to the chelate complexes [Cp₂Mo(urd-O2′,O3′)], [Cp₂Mo(ade-O2′,O3′)], [Cp₂Mo(cyd-O2′,O3′)], and [Cp₂Mo(gua-O2′,O3′)]. The ribonucleotides form three types of complex with Cp₂Mo²⁺ in neutral solution, namely N,PO-macrochelates, PO,O3′-coordinated species as well as O2′,O3′-chelates, while at pD 9 only sugar coordination is observed.     

Platinum(II) triammine antitumour complexes: structure-activity relationship with guanosine 5′-monophosphate (5′-GMP)

The multinuclear (¹H, ¹⁵N, ³¹P and ¹⁹⁵Pt) NMR spectroscopies, ES-MS and HPLC have been employed to investigate the structure-activity relationship for the reactions between guanosine 5′-monophosphate (5′-GMP) and the platinum(II)-triamine complexes of the general formulation cis-[Pt(NH₃)₂(Am)Cl]NO₃ (where Am represents a substituted pyridine). The order of reaction rate of the reactions was found to be: 3-phpy > 4-phpy > py > 4-mepy > 3-mepy > 2-mepy. The two basic factors, steric and electronic, were attributed to the order of the binding rate constants. A possible mechanism of the reaction of cis-[Pt(NH₃)₂(Am)Cl]⁺ with 5′-GMP suggested that the reactions proceed via direct nucleophilic attack and no loss of ammonia. cis-[Pt(NH₃)₂(Am)Cl]⁺ binds to the N7 nitrogen of the guanine residue of 5′-GMP to form a coordinate bond with the Pt metal centre. This mechanism is apparently different from that of cisplatin. The pK_a value of cis-[Pt(NH₃)₂(4-mepy)(H₂O)](NO₃)₂ (5.63) has been determined at 298 K by the use of distortionless enhancement by polarization transfer (DEPT) ¹⁵N NMR spectroscopy and compared to the pK_a value of cis-[PtCl(H₂O)(NH₃)₂]⁺.     

Purification and structural investigation of a water-soluble polysaccharide from Flammulina velutipes

A novel water-soluble heteropolysaccharide FVP60-B was extracted from the fruiting bodies of Flammulina velutipes with boiling water and purified by Sephacryl S-300 and S-400, which molecular weight was estimated to be 1.3 × 10⁴ Da by HPLC. It is composed of l-fucose, d-mannose, d-glucose and d-galactose in a ratio of 1.16:0.82:1.00:3.08. Sugar analysis, methylation analysis together with ¹H, ¹³C and 2D NMR spectroscopy disclosed that FVP60-B is consisted of a α-(1 → 6)-d-galactopyranan backbone with a terminal fucosyl, terminal glucosyl and α-(1 → 6)-d-mannopyranan units on O-2 of 2,6-O-substituted-d-galactosyl units.     

Mouse embryos fail to synthesize detectable quantities of guanosine 5'-diphosphate 3'-diphosphate.

The unusual highly phosphorylated nucleotide, guanosine 5′-diphosphate 3′-diphosphate, has been implicated in the control of development of the mouse (Irr, J. D., et al. (1974) Cell3, 249). We have been unable, however, to detect guanosine 5′-diphosphate 3′-diphosphate synthesis either in preimplantation and postimplantation mouse embryos cultured in the presence of [³²P]orthophosphate or in assays using ribosomes isolated from 10- to 13-day mouse embryos. Three unidentified phosphorous-containing compounds were detected in blastocyst stage mouse embryos.     

The synthesis of guanosine 5′-diphosphate-l-galactose by extracts of Chlorella pyrenoidosa

Extracts of the green alga Chlorella pyrenoidosa have been shown to catalyze the epimerization of guanosine 5′-diphosphate-d-mannose to guanosine 5′-diphosphate-l-galactose. The equilibrium is about 0.1 in the direction of the l-galactosyl nucleotide and is independent of temperature. The K_m for guanosine 5′-diphosphate-d-mannose was determined to be about 1.2 × 10^?4m. Guanosine 5′-diphosphate-l-fucose (6-deoxy-l-galactose) also serves as a substrate for the enzyme, and the product of that reaction appears to be guanosine 5′-diphosphate-d-rhamnose (6-deoxy-d-mannose).     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

Evolution of Class-Specific Peptides Targeting a Hot Spot of the Gαs Subunit

The four classes of heterotrimeric G-protein α subunits act as molecular routers inside cells, gating signals based on a bound guanosine nucleotide (guanosine 5′-triphosphate versus guanosine 5′-diphosphate). Ligands that specifically target individual subunits provide new tools for monitoring and modulating these networks, but are challenging to design due to the high sequence homology and structural plasticity of the Gα-binding surface. Here we have created an mRNA display library of peptides based on the short Gα-modulating peptide R6A-1 and selected variants that target a convergent protein-binding surface of Gαs·guanosine 5′-diphosphate. After selection/evolution, the most Gαs-specific peptide, Gαs(s)-binding peptide (GSP), was used to design a second-generation library, resulting in several new affinity- and selectivity-matured peptides denoted as mGSPs. The two-step evolutionary walk from R6A-1 to mGSP-1 resulted in an 8000-fold inversion in binding specificity, altered seven out of nine residues in the starting peptide core, and incorporated both positive and negative design steps. The resulting mGSP-1 peptide shows remarkable selectivity and affinity, exhibiting little or no binding to nine homologous Gα subunits or human H-Ras, and even discriminates the Gαs splice variant Gαs(l). Selected peptides make specific contacts with the effector-binding region of Gα, which may explain an interesting bifunctional activity observed in GSP. Overall, our work demonstrates a design of simple, linear, highly specific peptides that target a protein-binding surface of Gαs and argues that mRNA display-based selection/evolution is a powerful route for targeting protein families with high class specificity and state specificity.     

d-Mannose as a component of the macrophage surface receptor for macrophage-activating factor (MAF) in mice

Mouse peritoneal exudate macrophages were rendered cytostatic and cytolytic for various mouse tumor cells in vitro by exposure to partially purified lymphokines containing macrophage-activating factor (MAF) at 37 °C for 2 hr. The macrophage activation disappeared completely when either 0.1 Md-mannose or 0.1 M methyl-d-mannoside was present with MAF. On the other hand, neither d-galactose nor d-glucose inhibited the activation, and l-fucose, l-rhamnose, and N-acetyl-d-glucosamine inhibited it only partially. Incubation of either macrophages or MAF with 0.1 Md-mannose for 2 hr had no effect on activation of the macrophages by the MAF. Treatment of the macrophages by α-d-mannosidase rendered them no longer responsive to MAF. Macrophages treated by either neuraminidase or proteolytic enzymes, but not with β-d-galactosidase lost their ability to respond to MAF. Treatment of MAF with α-d-mannosidase did not affect MAF activity. In addition, MAF activity was not reduced by passage through a column of immobilized concanavalin A. In an absorption experiment, the presence of d-mannose was shown to prevent the adsorption of MAF to macrophages, while d-galactose did not. Treatment of macrophages with plant lectins having affinity for d-mannose, sialic acid or l-fucose prevented the adsorption of MAF, but the other lectins did not. Mouse MAF failed to adsorb to guinea pig peritoneal exudate macrophage, which were suggested as having a fucose-containing glycolipid as a lymphokine receptor. Taken together, these results strongly suggest that the receptor for MAF on mouse macrophages may be a glycoprotein containing d-mannose and sialic acid as essential components.     

Thermophilic l-fucose isomerase from Thermanaeromonas toyohensis for l-fucose synthesis from l-fuculose

The beneficial biological properties of l-fucose have extended its commercial application potential in pharmaceutical, cosmetic, and food industries. Enzymatic production of l-fucose with l-fucose isomerase (l-FucI) is considered a selective, green, and efficient strategy. Efficient sugar production requires thermophilic enzymes with increased reaction rate, reduced risk of microbial contamination, and high sugar solubility. No study has evaluated the applicability of thermophilic l-FucI for l-fucose production. In this study, we explored the biochemical properties of a thermostable l-FucI from Thermanaeromonas toyohensis (TtFucI) using l-fuculose as a substrate. The recombinant TtFucI exhibited thermophilicity and optimum activity at 70 °C. The specific activity, K_m, and k_cat toward l-fuculose were 199.8 U/mg, 33.4 mM, and 901.7 s⁻¹, respectively. Mn²⁺ ions increased the activity of the enzyme by ∼10 times and enhanced its thermal stability. Our study, on l-fucose synthesis by thermostable l-FucI, suggests the potential application of this enzyme for the industrial production of l-fucose.     

Pyruvate Kinase of Streptococcus lactis

The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5′-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic effector fructose-1,6-diphosphate, giving a typical Michaelis-Menten relationship. Increasing the concentration of fructose-1,6-diphosphate increased the apparent V_max values and decreased the K_m values for both substrates. Catalysis by pyruvate kinase proceeded optimally at pH 6.9 to 7.5 and was markedly inhibited by inorganic phosphate and sulfate ions. Under certain conditions adenosine 5′-triphosphate also caused inhibition. The K_m values for phosphoenolpyruvate and adenosine 5′-diphosphate in the presence of 2 mM fructose-1,6-diphosphate were 0.17 mM and 1 mM, respectively. The concentration of fructose-1,6-diphosphate giving one-half maximal velocity with 2 mM phosphoenolpyruvate and 5 mM adenosine 5′-diphosphate was 0.07 mM. The intracellular concentrations of these metabolites (0.8 mM phosphoenolpyruvate, 2.4 mM adenosine 5′-diphosphate, and 18 mM fructose-1,6-diphosphate) suggest that the pyruvate kinase in S. lactis approaches maximal activity in exponentially growing cells. The role of pyruvate kinase in the regulation of the glycolytic pathway in lactic streptococci is discussed.