Revision of the pathway of d-pinitol formation in leguminosae

Tracer studies with labelled inositols demonstrate that in contrast to earlier reports the formation of d-pinitol in Medicago sativa, Ononis spinosa and Trifolium incarnatum does not proceed by epimerization of sequoyitol but via d-ononitol. Hence, the novel pathway detected in Simmondsia chinensis occurs also in the Leguminous family and thus represents a major biosynthetic pathway for d-pinitol.     

D-chiro-inositol affects accumulation of raffinose family oligosaccharides in developing embryos of Pisum sativum

Developing garden pea embryos are able to take up exogenously applied cyclitols: myo-inositol, which naturally occurs in pea, and two cyclitols absent in pea plants: d-chiro-inositol and d-pinitol. The competition in the uptake of cyclitols by pea embryo, insensitivity to glucose and sucrose, and susceptibility to inhibitor(s) of H⁺-symporters (e.g. CCCP and antimycin A) suggest that a common cyclitol transporter is involved. Both d-chiro-inositol and d-pinitol can be translocated through the pea plant to developing embryos. During seed maturation drying, they are used for synthesis of mainly mono-galactosides, such as fagopyritol B1 and galactosyl pinitol A. Accumulation of d-chiro-inositol (and formation of fagopyritols), but not d-pinitol, strongly reduces accumulation of verbascose, the main raffinose oligosaccharide in pea seeds. The reasons for the observed changes are discussed.     

Studies on l-ascorbic acid biosynthesis and metabolism in Parthenocissus quinquefolia L. (vitaceae)

Parthenocissus quinquefolia (L.) Planch., commonly known as Virginia Creeper, is a vitaceous tartrate-accumulating vine that exhibits C-4/C-5 cleavage of l-ascorbic acid (AA) to produce l-tartaric acid (TA) from the C₄ fragment and carbohydrate pool material from the C₂ fragment. Experiments in which detached leaves were supplied d-[5-³H,1-¹⁴C]glucose or d-[5-³H,6-¹⁴C]glucose yielded AA devoid of ³H whereas the l-threonic acid (ThA) and TA recovered from the same tissues still retained some ³H. These comparative experiments also indicated that the ThA was derived from carbons 3 through 6 of d-glucose. ThA was shown to be a natural constituent of P. quinquefolia but apparently not an intermediate between AA and TA. Results are consistent with a biosynthetic pathway from d-glucose to AA that involves a hydrogen-exchanging epimerization at C-5 as reported earlier for the geraniaceous plant Pelargonium crispum, but differing from P.crispum in biosynthesis and metabolism of ThA.When l-[6-¹⁴C]idonate or its lactone was supplied to P. quinquefolia leaves, about 80% of the ¹⁴C appeared in the carbohydrates, an observation remarkably similar to previous observations with [6-¹⁴C]AA-labeled leaves. l-Idonate and its lactone appear to have an intermediate role in AA metabolism in vitaceous plants.     

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).     

The epimerization of chiral half sandwich 2-phenylpyridine-based ruthenacycle

The reaction of a chiral terpenic 2-phenylpyridine with mercury (II) acetate afforded a new ortho-chloromercurated substrate, which was treated with [(η⁶-cymene)RuCl₂]₂ to afford two diastereomers of the corresponding half-sandwich cyclochlororuthenated terpenic ligand. The latter two complexes were successfully separated by conventional chromatography and their configurational stability investigated by ¹H NMR and CD spectroscopy. It was found that both isolated diastereomers displayed a fair configurational stability in CH₂Cl₂ while when dissolved in MeOH they epimerize. It was shown that the rate of epimerization is rather slow in pure MeOH, whereas in the presence of an excess of Lewis acid such as HgCl₂ it was notably accelerated suggesting that the decoordination of the chloride from the Ru(II) center is a key step in the epimerization process.     

S-adenosylmethionine: Stability and stabilization

With a developed HPLC technique for the separation of both (+)- and (−)-S-adenosylmethionine (AdoMet) and ¹H NMR analysis of the epimeric S-CH₃ chemical shifts, a kinetic study on the stability of (−)-AdoMet in solution to decomposition and epimerization is described. The results obtained from the effects of pH, temperature, and sulfonium counterions on the stability of AdoMet indicate that the epimerization appears to proceed through pyramidal inversion of the sulfonium pole. The optimal conditions for AdoMet to be stable in solution to decomposition and epimerization is to keep the compound at pH 3–5, containing an excess of large-size, nonnucleophilic counterions such as tosylate or sulfate.     

Formation of l-quebrachitol from d-bornesitol in leaves of Acer pseudoplatanus

d-Bornesitol and l-quebrachitol have been found in the leaves of Acer pseudoplatanus L. The results of incorporation studies using labeled myo-inositol-¹⁴C, l-inositol-¹⁴C and d-bornesitol-¹⁴C indicate that l-quebrachitol is produced by epimerization of d-bornesitol. In Artemisia vulgaris, however, the precursor of l-quebrachitol is l-inositol.     

The synthesis of guanosine 5′-diphosphate l-fucose by enzymes of a higher plant

An enzyme preparation from the leaves of Phaseolus vulgaris catalyzes the reduction and epimerization of guanosine 5′-diphosphate d-mannose to guanosine 5′-diphosphate l-fucose. Ethylenediaminetetraacetic acid must be added to the reaction mixtures to prevent the enzymic breakdown of the product to a compound presumed to be l-fucose 1-phosphate. The synthetic reaction requires NADPH, has a pH optimum between 7 and 8 an apparent K_m for guanosine 5′-diphosphate d-mannose of 1.6·10⁻⁴ M. Evidence is presented that guanosine 5′-diphosphate 4-keto-6-deoxy-d-mannose is an intermediate in the reaction.     

Synthesis of 1α,25-dihydroxyvitamin D2, its 24 epimer and related isomers,and their binding affinity for the 1,25-dihydroxyvitamin D3 receptor

The synthesis of 1α-25-dihydroxyvitamin D₂ and of several stereoisomers (5,6-trans and 1β-hydroxy isomers and the 24R-epimers of these compounds) was reported. Synthesis was accomplished from two different starting materials, 25-hydroxyvitamin D₂ and 25,25-ethylenedioxy-26-norvitamin D₂, and involved C-1-hydroxylation via 3,5-cyclovitamin D intermediates. Synthetic 1α,25-dihydroxyvitamin D₂ was found to be identical with the biologically generated natural product. An analysis of the binding affinity of the synthetic products for the 1α,25-dihydroxyvitamin D₃ receptor protein showed that isomerization of the 5,6 double bond from cis to trans, or epimerization of the 24-methyl group from S to R, reduced ligand binding to the receptor only slightly, while both changes together led to a 100-fold reduction of binding affinity. The epimerization of the 1-hydroxy function from 1α to 1β attenuated binding dramatically (ca. 1000-fold).     

The alpha beta epimerization of reduced nicotinamide adenine dinucleotide.

The proton magnetic resonance spectra of the dihydronicotinamide ring of αNADH³ and the nicotinamide ring of αNAD⁺ are reported and the proton absorptions assigned. The absolute assignment of the C4 methylene protons of αNADH is based on the generation of specifically deuterium-labeled (pro-S) B-deuterio-αNADH from enzymatically prepared B-deuterio-βNADH. The C4 proton absorption of αNAD⁺ is assigned by oxidation of B-deuterio-αNADH by the A specific, yeast alcohol dehydrogenase to yield 4-deuterio-αNAD⁺.The epimerization of either αNADH or βNADH yields an equilibrium ratio of approximately 9:1 βNADH to αNADH. The rate of epimerization of αNADH to βNADH at 38 °C in 0.05, pH 7.5, phosphate buffer is 3.1 × 10^?3 min^?1, corresponding to a half-life of 4 hr. Four related dehydrogenases, yeast and horse liver alcohol dehydrogenase and chicken M₄ and H₄ lactate dehydrogenase, are shown to oxidize αNADH to αNAD⁺ at rates three to four orders of magnitude slower than for βNADH. By using specifically labeled B-deuterio-αNADH the enzymatic oxidation by yeast alcohol dehydrogenase has been shown to occur with the identical stereospecificity as the oxidation of βNADH. The nonenzymatic epimerization of αNADH to βNADH and the enzymatic oxidation αNADH are discussed as a possible source of αNAD⁺in vivo.     

Chain elongation by a seemingly stereospecific cyanohydrin synthesis: the preparation and configurational assignment of 3,7-anhydro-d-threo-l-talo- and -l-galacto-octose

2,6-Anhydro-d-glycero-l-manno-heptose (1) is converted by the cyanohydrin reaction into crystalline d-threo-l-talo-octononitrile (3), which shows mutarotation in water. The equilibrium mixture, as measured by ¹³C-n.m.r. spectroscopy, contains about equal amounts of 3 and its epimer, d-threo-l-galacto-octononitrile. On evaporation of the aqueous mixture, pure, crystalline 3 is again obtained. Labelling experiments in ³H₂O proved that epimerization proceeds through reversible deprotonation. Stabilization of 3 in the solid state is explained by intramolecular hydrogen-bonding. In pyridine, rapid isomerization of 3 occurs. When acetylation of 3 is conducted in this solvent, the yield of 2,4,5,6,8-penta-O-acetyl-3,7-anhydro-d-threo-l-talo-octono-nitrile (4) depends strongly on the conditions of acetylation. Acetylation after equilibration produces an equimolar mixture of 4 and its isomer 2,4,5,6,8-penta-O-acetyl-3,7-anhydro-d-threo-l-galacto-octononitrile. Structural assignment for both was achieved by 360-Mhz, ¹H- and ¹³C-n.m.r. spectroscopy. Reduction of 4 in pyridine-acetic acid-water in the presence of N,N-diphenylethylenediamine yields a 1:2.36 mixture of 2,4,5,6,8-penta-O-acetyl-3,7-anhydro-d-threo-l-talo-octose N,N-diphenylimidazolidine (6) and 2,4,5,6,8-penta-O-acetyl-3,7-anhydro-d-threo-l-galacto-octose N,N-diphenylimidazolidine (8). Compounds 6 and 8 could be separated and obtained as crystalline solids, and their structure proved by ¹H- and ¹³C-n.m.r. spectroscopy. Hydrolysis of 6 and 8 gave 2,4,5,6,8-penta-O-acetyl-3,7-anhydro-d-threo-l-galacto-octose and -d-threo-l-talo-octose.     

Energy Landscape of Alginate-Epimerase Interactions Assessed by Optical Tweezers and Atomic Force Microscopy

Mannuronan C-5 epimerases are a family of enzymes that catalyze epimerization of alginates at the polymer level. This group of enzymes thus enables the tailor-making of various alginate residue sequences to attain various functional properties, e.g. viscosity, gelation and ion binding. Here, the interactions between epimerases AlgE4 and AlgE6 and alginate substrates as well as epimerization products were determined. The interactions of the various epimerase–polysaccharide pairs were determined over an extended range of force loading rates by the combined use of optical tweezers and atomic force microscopy. When studying systems that in nature are not subjected to external forces the access to observations obtained at low loading rates, as provided by optical tweezers, is a great advantage since the low loading rate region for these systems reflect the properties of the rate limiting energy barrier. The AlgE epimerases have a modular structure comprising both A and R modules, and the role of each of these modules in the epimerization process were examined through studies of the A- module of AlgE6, AlgE6A. Dynamic strength spectra obtained through combination of atomic force microscopy and the optical tweezers revealed the existence of two energy barriers in the alginate-epimerase complexes, of which one was not revealed in previous AFM based studies of these complexes. Furthermore, based on these spectra estimates of the locations of energy transition states (x _β), lifetimes in the absence of external perturbation (τ ⁰) and free energies (ΔG ^#) were determined for the different epimerase–alginate complexes. This is the first determination of ΔG ^# for these complexes. The values determined were up to 8 k_BT for the outer barrier, and smaller values for the inner barriers. The size of the free energies determined are consistent with the interpretation that the enzyme and substrate are thus not tightly locked at all times but are able to relocate. Together with the observed different affinities determined for AlgE4-polymannuronic acid (poly-M) and AlgE4-polyalternating alginate (poly-MG) macromolecular pairs these data give important contribution to the growing understanding of the mechanisms underlying the processive mode of these enzymes.     

Comparison of the genetic activity of aflatoxins B1 and G1 in Escherichia coli and Saccharomyces cerevisiae

The ability of aflatoxins B₁ and G₁ to induce back mutations to arg⁺ in Escherichia coli K-12/343/113 was compared with induction of mitotic gene conversion to ade⁺ in the diploid yeast strain Saccharomyces cerevisiae D4, ade₂^?. In analogy to previous results with other microorganisms, the compounds were not genetically active per se, indicating that under the experimental conditions employed none of the tester strains were able to activate the compounds to mutagenic products.In experiments using liver homegenates (S-9 fraction) of male Golden Syrian hamsters previously treated with phenobarbital, aflatoxin B₁ exhibited strong genetic activity both in E. coli and in S. cerevisiae, whereas the mutagenic activity of aflatoxin G₁ was markedly lower and could be detected only in the E. coli tester strain. These results correlate the findings that aflatoxin G₁ is a less potent carcinogen and mutagen than aflatoxin B₁.     

l-Ribose from l-arabinose by epimerization and its purification by 3-zone simulated moving bed chromatography

l-Ribose has recently received attention as the starting material for nucleoside drugs. As it is not found in nature, it is being produced by enzymatic or epimerization reaction. We investigated an epimerization reaction by molybdenium oxide and examined the effects of temperature, solvent, and molybdenum oxide amount on epimerization. l-Ribose has a yield of 22% under the conditions of 100 kg/m³ l-arabinose, 20% methanol, 5 kg/m³ MoO₃, and 90°C. In addition, simulated moving bed (SMB) that was equipped with three NH₂-HPLC columns was used to separate l-arabinose and l-ribose resulting from l-arabinose epimerization. A 3-zone SMB process was developed to eliminate the high pressure problem in the conventional 4-zone SMB. Aspen simulation was performed to determine the operating variables such as switching time, raffinate, and extract flow rates. Experimental purities of extract and raffinate were compared with the theoretical ones and they are found to be fairly well correlated.     

Determination of the hydraulic conductivity and of reflection coefficients in Nitella flexilis by means of direct cell-turgor pressure measurements

From direct and continuous measurements of the internal hydrostatic pressure (P) in the internodes of Nitella flexilis, the reflection coefficients (σ_s) of some non-electrolytes were determined, using a zero-flow method, and were compared with those found previously on Valonia utricularis and with those obtained by Dainty and Ginzburg on other Characean internodes from transcellular osmosis experiments. The hydraulic conductivities (Lp) of the cell membranes were determined by two independent methods, that is, using hydrostatically or osmotically induced flows. From the exponential time course of P in such experiments and from the volumetric elastic modulus (ε) of the cell wall, Lp was calculated. The effect of unstirred layers in the methods described was negligibly small.In osmotic experiments with different non-plasmolysing external sucrose concentrations (20–200 mM) the exosmotic hydraulic conductivity (Lp_ex) decreases markedly with increasing concentration, while the endosmotic hydraulic conductivity (Lp_en) shows only a weak dependence. In the hydrostatic experiments the hydraulic conductivities for single cells were constant in the pressure range for P from 2 to 7 atm. In this pressure range Lp_en and Lp_ex varied for different cells from 2.2·10^?5 to 2.8·10^?5 and from 1.8·10^?5 to 2.5·10^?5 cm·s^?1·atm^?1, respectively, with an average ratio Lp_en to Lp_ex of 1.1, which indicates a polarity in water movement.These values were the same as those obtained in the osmotic experiments from extrapolation to zero sucrose concentration. At internal pressures below 2 atm the Lp-values markedly increase on approaching the plasmolytic point.The results are discussed in terms of a dehydration of the membranes (or the cytoplasm) at increased solute concentrations. In addition, the strong dependence of Lp at low internal hydrostatic pressures points to a direct influence of P on the water permeability of the membranes.     

Sodium-dependent isoleucine transport in the methanogenic archaebacterium Methanococcus voltae

Methanococcus voltae possesses a Na⁺-dependent transport system for isoleucine which requires for optimum rates a CO₂/H₂ atmosphere. The K_m for the system is 4.5 μM with a V_max of 1.5 nmol·min^?1·mg dry wt^?1. Approximately 75% of the label can be released from the cell pool following short-term experiments with gradients of isoleucine reaching 100 (in/out). Transport is inhibited by ionophores and N-ethyl maleimide. Only valine and leucine effectively compete with isoleucine for transport.     

The Variation of Root Exudates from the Hyperaccumulator Sedum alfredii under Cadmium Stress: Metabonomics Analysis

Hydroponic experiments were conducted to investigate the variation of root exudates from the hyperaccumulator Sedum alfredii under the stress of cadmium (Cd). S. alfredii was cultured for 4 days in the nutrient solution spiked with CdCl₂ at concentrations of 0, 5, 10, 40, and 400 µM Cd after the pre-culture. The root exudates were collected and analyzed by GC-MS, and 62 compounds were identified. Of these compounds, the orthogonal partial least-squares discrimination analysis (OPLS-DA) showed that there were a distinct difference among the root exudates with different Cd treatments and 20 compounds resulting in this difference were found out. Changing tendencies in the relative content of these 20 compounds under the different Cd treatments were analyzed. These results indicated that trehalose, erythritol, naphthalene, d-pinitol and n-octacosane might be closely related to the Cd stabilization, phosphoric acid, tetradecanoic acid, oxalic acid, threonic acid and glycine could be attributed to the Cd mobilization, and mannitol, oleic acid, 3-hydroxybutanoic acid, fructose, octacosanol and ribitol could copy well with the Cd stress.     

Characterization and mutational analysis of two UDP-galactose 4-epimerases in <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> TIGR4

Current clinical treatments for pneumococcal infections have many limitations and are faced with many challenges. New capsular polysaccharide structures must be explored to cope with diseases caused by different serotypes of Streptococcus pneumoniae. UDP-galactose 4-epimerase (GalE) is an essential enzyme involved in polysaccharide synthesis. It is an important virulence factor in many bacterial pathogens. In this study, we found that two genes (galE _sp1 and galE _sp2 ) are responsible for galactose metabolism in pathogenic S. pneumoniae TIGR4. Both GalE_Sp1 and Gal_ESp2 were shown to catalyze the epimerization of UDP-glucose (UDP-Glc)/UDP-galactose (UDP-Gal), but only GalE^Sp2 was shown to catalyze the epimerization of UDP-N-acetylglucosamine (UDP-GlcNAc)/UDP-N-acetylgalactosamine (UDP-GalNAc). Interestingly, GalE_Sp2 had 3-fold higher epimerase activity toward UDP-Glc/UDP-Gal than GalE_Sp1. The biochemical properties of GalE_Sp2 were studied. GalE_Sp2 was stable over a wide range of temperatures, between 30 and 70°C, at pH 8.0. The K86G substitution caused GalE_Sp2 to lose its epimerase activity toward UDP-Glc and UDP-Gal; however, substitution C300Y in GalE_Sp2 resulted in only decreased activity toward UDP-GlcNAc and UDP-GalNAc. These results indicate that the Lys86 residue plays a critical role in the activity and substrate specificity of GalE_Sp2.     

Conformational and Functional Effects Induced by D- and L-Amino Acid Epimerization on a Single Gene Encoded Peptide from the Skin Secretion of Hypsiboas punctatus

Skin secretion of Hypsiboas punctatus is the source of a complex mixture of bioactive compounds where peptides and small proteins prevail, similarly to many other amphibians. Among dozens of molecules isolated from H. punctatus in a proteomic based approach, we report here the structural and functional studies of a novel peptide named Phenylseptin (FFFDTLKNLAGKVIGALT-NH₂) that was purified as two naturally occurring D- and L-Phes configurations. The amino acid epimerization and C-terminal amidation for both molecules were confirmed by a combination of techniques including reverse-phase UFLC, ion mobility mass spectrometry, high resolution MS/MS experiments, Edman degradation, cDNA sequencing and solid-phase peptide synthesis. RMSD analysis of the twenty lowest-energy ¹H NMR structures of each peptide revealed a major 90° difference between the two backbones at the first four N-terminal residues and substantial orientation changes of their respective side chains. These structural divergences were considered to be the primary cause of the in vitro quantitative differences in antimicrobial activities between the two molecules. Finally, both molecules elicited equally aversive reactions in mice when delivered orally, an effect that depended entirely on peripheral gustatory pathways.