Cyclitols affect accumulation of α-<Emphasis Type="SmallCaps">d</Emphasis>-galactosides in developing <Emphasis Type="Italic">Vicia</Emphasis> seeds

The mechanism preferentially regulating accumulation of raffinose family oligosaccharides (RFOs) or galactosyl cyclitols in legume seeds still remains unknown. The broad range of raffinose family oligosaccharides and galactosyl pinitols in the composition of seeds of Vicia genus gives researchers an exceptional opportunity for investigations on relationships in biosynthesis of both types of α-d-galactosides. Feeding explants of Vicia species radically different in the composition of RFOs and galactosyl pinitols with basic galactose acceptors, sucrose (for RFOs) or cyclitols (for galactosyl cyclitols) can be a helpful method for assessment of their regulatory role in accumulation of α-d-galactosides in seeds. Garden vetch (Vicia sativa L.) seeds, naturally accumulating RFOs, demonstrated an ability to take up and use exogenously applied d-pinitol and d-chiro-inositol for synthesis of their mono-, di- and tri-galactosides. Together with the accumulation of new galactosides, the concentration of RFOs decreased. In fine-leaved (Vicia tenuifolia Roth.) vetch seeds such a remarkably high concentration of galactosyl pinitols (GPs) was discovered that they nearly replaced RFOs, which is unique among legumes. If the accumulation of both types of galactosides is correlated with concentration of galactose acceptors, elevated levels of sucrose or myo-inositol should promote accumulation of RFOs, instead of GPs. Unexpectedly, feeding fine-leaved vetch raceme explants with myo-inositol or sucrose promoted accumulation of GPs, but not of RFOs. Our comparison of accumulation and biosynthesis of both types of galactosides (RFOs and GPs) throughout development and maturation of seeds from fine-leaved vetch has indicated that preferential accumulation of GPs is associated with the drying of seeds during maturation. Different patterns in activities of enzymes engaged in RFOs’ biosynthetic pathway and galactosyltransferases involved in biosynthesis of GPs indicated that distinct forms of enzymes can operate in both pathways. The feeding of explants with d-chiro-inositol causes accumulation of fagopyritols B1 in seeds of both Vicia species, which suggests presence of the same or a similar form of galactinol synthase. Accumulation of fagopyritols in fine-leaved vetch seeds did not affect accumulation of RFOs or galactosyl pinitols.     

Rat L6 myotubes as an in vitro model system to study GLUT4-dependent glucose uptake stimulated by inositol derivatives

Some of inositol derivatives have been reported to help the action of insulin stimulating glucose uptake in skeletal muscle cells. Rat L6 myotubes were employed in an attempt to develop an in vitro model system for investigation of the possible insulin-like effect of eight inositol derivatives, namely allo-inositol, d-chiro-inositol l-chiro-inositol, epi-inositol, muco-inositol, myo-inositol, scyllo-inositol and d-pinitol. At a higher concentration of 1 mM seven inositol derivatives other than myo-inositol were able to stimulate glucose uptake, while at 0.1 mM only d-chiro-inositol, l-chiro-inositol, epi-inositol and muco-inositol could induce glucose uptake, indicating their significant insulin-mimetic activity. Immunoblot analyses revealed that at least d-chiro-inositol, l-chiro-inositol, epi-inositol, muco-inositol and d-pinitol were able to induce translocation of glucose transporter 4 (GLUT4) to plasma membrane not only in L6 myotubes but also in skeletal muscles of rats ex vivo. These results demonstrated that L6 myotubes appeared efficient as an in vitro system to identify inositol derivatives exerting an insulin-like effect on muscle cells depending on the induced translocation of GLUT4.     

Roles of Ile66 and Ala107 of d-psicose 3-epimerase from Agrobacterium tumefaciens in binding O6 of its substrate, d-fructose

Using site-directed mutagenesis, we investigated the roles of Ile66 and Ala107 of d-psicose 3-epimerase from Agrobacterium tumefaciens in binding O6 of its true substrate, d-fructose. When Ile66 was substituted with alanine, glycine, cysteine, leucine, phenylalanine, tryptophan, tyrosine or valine, all the mutants dramatically increased the K _m for d-tagatose but slightly decreased the K _m for d-fructose, indicating that Ile66 is involved in substrate recognition. When Ala107 was substituted by either isoleucine or valine, the substituted mutants had lower thermostability than the wild-type enzyme whereas the proline-substituted mutant had higher thermostability. Thus, Ala107 is involved in enzyme stability.     

Differences in accumulation of soluble α-galactosides during seed maturation of several <Emphasis Type="Italic">Vicia</Emphasis> species

Composition and levels of soluble α-galactosides: raffinose family oligosaccharides (RFOs) and galactosyl cyclitols (Gal-C) in developing seeds were measured by high resolution gas chromatography (HRGC) method. The studies were performed on maturing seeds of several wild and cultivated Vicia species: Vicia angustifolia L. (common vetch), Vicia cracca L. (bird vetch), Vicia grandiflora Scop. (large yellow vetch), Vicia hirsuta (L.) S.F.Gray (tiny vetch), Vicia sativa L. (garden vetch, spring-growing cultivar Kwarta), and Vicia villosa Roth (winter vetch). In all Vicia species similar patterns in the accumulation of RFOs were observed. Galactinol — the donor of galactosyl moieties in α-galactosides biosynthesis was present in the middle stage of seed development, before appearing measurable levels of RFOs. Accumulation of RFOs started parallel with seed desiccation process. At first accumulation of the raffinose, then few days later stachyose and finally verbascose was noticed. In the final stage of seed maturation the verbascose was the main soluble α-galactoside (up to 3% of dry weight, V. sativa). Besides the RFOs seeds of three Vicia species (V. cracca, V. hirsuta, and V. villosa) accumulated d-pinitol and its α-galactosides (Gal-C). Mono-galactosylpinitols (similar to raffinose) appeared in these species 2–4 days after galactinol, di-galactosyl pinitol A (common name: ciceritol) and di-galactosyl myo-inositol were present several days later than raffinose, and accumulation of tri-galactosyl pinitol A (TGPA) began after accumulation of stachyose. Matured seeds of V. hirsuta contained much more RFOs than Gal-C, opposite to seeds of V. villosa, and V. cracca where concentration of Gal-C was 4–8-fold higher than RFOs. In V. cracca seeds RFOs were almost replaced by Gal-C. In seeds of V. cracca and V. villosa the level of d-pinitol was significantly higher, than the level of myo-inositol. Contents of both cyclitols declined rapidly at the beginning of seed desiccation, when accumulation of RFOs and Gal-C quickly increased. We suggest that α-galactosides of d-pinitol can substitute raffinose family oligosaccharides and play similar role during seed maturation and storage.     

Determination of two sites of automethylation in bovine erythrocyte protein (d -aspartyl/l -isoaspartyl) carboxyl methyltransferase

Protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM, E.C. 2.1.1.77) was previously shown to be enzymatically methyl esterified in an autocatalytic manner at altered aspartyl residues; methyl esters are observed in a subpopulation of the enzyme termed thePCM fraction [Lindquist and McFadden (1994),J. Protein Chem. 13, 23–30]. The altered aspartyl sites serving as methyl acceptors inPCM have now been localized by using proteolytic enzymes and chemical cleavage techniques in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify fragments of the [³H]automethylated enzyme that contain a [³H]methyl ester. Methylation was positively identified at positions Asn₁₈₈ and Asp₂₁₇ in the enzyme sequence, a consequence of the spontaneous alteration of these sites tol-isoaspartyl ord-aspartyl sites and their methylation by active PCM molecules. The identification of more than one site of automethylation shows thatPCM is not a homogeneous population of damaged PCM molecules, but rather a complex population of molecules with a variety of age-altered damage sites.Abbreviations PCM protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase - EDTA disodium ethylenediaminetetraacetate - PMSF phenylmethylsulfonyl fluoride - TEA trifluoroacetic acid - HPLC high-pressure liquid chromatography     

Diastereoselective synthesis of l-threo-3,4-dihydroxyphenylserine by low-specific l-threonine aldolase mutants

Diastereoselectivity-enhanced mutants of l-threonine aldolase (l-TA) for l-threo-3,4-dihydroxyphenylserine (l-threo-DOPS) synthesis were isolated by error-prone PCR followed by a high-throughput screening. The most improved mutant was achieved from the mutant T3-3mm2, showing a 4-fold increase over the wild-type l-TA. When aldol condensation activity was examined using whole cells of T3-3mm2, its de was constantly maintained at 55% during the batch reactions for 80 h, yielding 3.8 mg l-threo-DOPS/ml.     

Automethylation of protein (d -aspartyl/l -isoaspartyl) carboxyl methyltransferase, a response to enzyme aging

A question that is central to understanding the mechanisms of aging and cellular deterioration is whether enzymes involved in recognition and metabolism of spontaneously damaged proteins are themselves damaged, either becoming substrates for their own activity; or being unable to act upon themselves, initiating cascades of cellular damage. We show here byin vitro experiments that protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM) from bovine erythrocytes does methylate age-dependent amino acid damage in its own sequence. The subpopulation that is methylated, termed thePCM fraction, appears to be formed through age-dependent deamidation of an asparaginyl site to either anl-isoaspartyl ord-aspartyl site because (a) the stoichiometry of automethylation of purified PCM is less than 1%, a value typical of the substoichiometric methylation of many other aged protein substrates, (b)PCM is slightly more acidic than the bulk of PCM, and (c) the methyl esterified site inPCM has the characteristic base-lability of this type of methyl ester. Also, the methyl group is not incorporated into the enzyme as an active site intermediate because the incorporated methyl group is not chased onto substrate protein. The effect of enzyme dilution on the rate of the automethylation reaction is consistent with methylation occurring between protein molecules, showing that the pool of PCM is autocatalytic even though individual molecules may not be. The automethylation and possible self-repair of the PCM pool has implications for maintaining thein vivo efficiency of methylation-dependent protein repair.     

Primary Structure, sequence analysis, and expression of the thermostable d - hydantoinase from Bacillus stearothermophilus SD1

The gene coding for the thermostable d-hydantoinase from the thermophilic bacterium Bacillus stearothermophilus SD1 was cloned and its nucleotide sequence was completely determined. The d-hydantoinase protein showed considerable amino acid sequence homology (20–28%) with other hydantoinases and functionally related allantoinases and dihydroorotases. Strikingly the sequence of the enzyme from B. stearothermophilus SD1 exhibited greater than 89% identity with hydantoinases from thermophilic bacteria. Despite the extremely high amino acid homology among the hydantoinases from thermophiles, the C-terminal regions of the enzymes were completely different in both sequence and predicted secondary structure, implying that the C-terminal region plays an important role in determining the biochemical properties of the enzymes. Alignment of the sequence of the d-hydantoinase from B. stearothermophilus SD1 with those of other functionally related enzymes revealed four conserved regions, and five histidines and an acidic residue were found to be conserved, suggesting a close evolutionary relationship between all these enzymes. Received: 20 December 1996 / Accepted: 12 March 1997     

In situ kinetic measurements of d -amino acid oxidase in rat liver with respect to its substrate specificity

Summary d-Amino acid oxidase activity was demonstrated in peroxisomes of rat liver using unfixed cryostat sections and a histochemical technique using cerium ions as capture reagent for hydrogen peroxide and diaminobenzidine, cobalt ions and exogenous hydrogen peroxide to visualize the final reaction product for light microscopical analysis. Cytophotometric analysis of liver sections revealed similar zero-order reaction velocities of d-amino acid oxidase with activity twice as high in periportal areas as in pericentral areas of liver lobuli when using either d-proline or d,l-thiazolidine-2-carboxylic acid as substrates. On the other hand, a 4–5 times higher K _M value was found for d-proline than for d,l-thiazolidine-2-carboxylic acid. The K _M values in periportal and pericentral areas were similar for each substrate. These findings support the suggestion that the physiological substrate for d-amino acid oxidase may be d,l-thiazolidine-2-carboxylic acid, the adduct of cysteamine and glyoxylic acid. d-Amino acid oxidase may play a role in vivo in the production of oxalate which may participate in metabolic control processes as an intracellular messenger molecule.     

Structural studies of neuropeptides composed ofl -Asp andd -Glu by13C-NMR spectroscopy

¹³C-NMR spectral data are presented for four neuropeptides composed ofl-Asp, Ac-l-Asp, andd-Glu, which contain and peptide linkages. The data for the various compounds are compared to related dipeptides, one of which is a known neuropeptide, in order to gain structural information about these compounds. Electron-nuclear relaxation rates were used to elucidate the metal-ion binding sites of these species.     

Raffinose in seedlings of winter vetch (<Emphasis Type="Italic">Vicia villosa</Emphasis> Roth.) under osmotic stress and followed by recovery

During germination of winter vetch (Vicia villosa Roth.) seeds, the degradation of raffinose family oligosaccharides and galactosyl pinitols occurred faster in axis than in cotyledons. After 7 days of germination, all α-d-galactosides disappeared and the soluble carbohydrates in seedling tissues consisted of d-pinitol, sucrose, fructose, glucose and myo-inositol. Osmotic stress caused by incubation of seedlings in PEG 8000 solution (−0.5, −1.0, and −1.5 MPa) for 48 h induced the activity of crucial enzymes of the RFOs pathway, i.e. galactinol synthase and raffinose synthase, in both the root and epicotyl but not in cotyledons. The root and epicotyl accumulated elevated amounts of galactinol and raffinose as the osmotic potential was lowered. This process was transient because when PEG solution was replaced with water, galactinol and raffinose were degraded, thus confirming their direct involvement in the response of tissues to osmotic stress. Among other soluble carbohydrates, only sucrose accumulated in response to stress. The results did not show potential role of d-pinitol in the adjustment of winter vetch seedlings to osmotic stress.     

The occurrence and accumulation of <Emphasis Type="SmallCaps">d</Emphasis>-pinitol in fenugreek (<Emphasis Type="Italic">Trigonella foenum graecum</Emphasis> L.)

This article presents changes in concentrations of d-pinitol (and other cyclitols as well as low molecular weight carbohydrates) in vegetative and reproductive organs of fenugreek (Trigonella foenumgraecum L.) during an entire plant growing period. d-Pinitol was the major cyclitol in all tested organs, representing 43–94% of total cyclitols and 2–77% of total soluble carbohydrates. The highest concentration of d-pinitol was found in pods (14–23 mg g^?1 of dry weight, DW), lower in leaves and stems (5–20 and 9–10 mg g^?1 DW, respectively), and the lowest in maturing seeds (2–5 mg g^?1 DW). Although maturing seeds accumulate α-d-galactosides of d-pinitol (galactosyl pinitols, up to 6.6 mg g^?1 DW), the major storage sugars were raffinose family oligosaccharides (RFOs, 65.37 mg g^?1 DW). Both RFOs and galactosyl pinitols are hydrolyzed during seed germination, releasing sucrose and d-pinitol, respectively. Accumulation of free galactose was not detected. Owing to the high concentration of d-pinitol (up to 23.70 mg g^?1 DW) and low concentration of soluble sugars, developing pods seem to be the best source of d-pinitol.     

Fagopyritol B1, O-α-D-galactopyranosyl-(1→2)-D-chiro-inositol, a galactosyl cyclitol in maturing buckwheat seeds associated with desiccation tolerance

O-α-D-Galactopyranosyl-(1→2)-D-chiro-inositol, herein named fagopyritol B1, was identified as a major soluble carbohydrate (40% of total) in buckwheat (Fagopyrum esculentum Moench, Polygonaceae) embryos. Analysis of hydrolysis products of purified compounds and of the crude extract led to the conclusion that buckwheat embryos have five α-galactosyl D-chiro-inositols: fagopyritol A1 and fagopyritol B1 (mono-galactosyl D-chiro-inositol isomers), fagopyritol A2 and fagopyritol B2 (di-galactosyl D-chiro-inositol isomers), and fagopyritol B3 (tri-galactosyl D-chiro-inositol). Other soluble carbohydrates analyzed by high-resolution gas chromatography included sucrose (42% of total), D-chiro-inositol, myo-inositol, galactinol, raffinose and stachyose (1% of total), but no reducing sugars. All fagopyritols were readily hydrolyzed by α-galactosidase (EC 3.2.1.22) from green coffee bean, demonstrating α-galactosyl linkage. Retention time of fagopyritol B1 was identical to the retention time of O-α-D-galactopyranosyl-(1→2)-D-chiro-inositol from soybean (Glycine max (L.) Merrill, Leguminosae), suggesting that the α-ga-lactosyl linkage is to the 2-position of D-chiro-inositol. Accumulation of fagopyritol B1 was associated with acquisition of desiccation tolerance during seed development and maturation in planta, and loss of fagopyritol B1 correlated with loss of desiccation tolerance during germination. Embryos of seeds grown at 18 °C, a condition that favors enhanced seed vigor and storability, had a sucrose-to-fagopyritol B1 ratio of 0.8 compared to a ratio of 2.46 for seeds grown at 25 °C. We propose that fagopyritol B1 facilitates desiccation tolerance and storability of buckwheat seeds. Received: 21 May 1997 / Accepted: 5 June 1997     

Structure of the O-antigen and characterization of the O-antigen gene cluster of Escherichia coli O108 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic (8-epilegionaminic) acid

On mild acid degradation of the lipopolysaccharide of Escherichia coli O108, the O-polysaccharide was isolated and studied by sugar analysis and one- and two-dimensional ¹H- and ¹³C-NMR spectroscopy. The polysaccharide was found to contain an unusual higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic acid (di-N-acetyl-8-epilegionaminic acid, 8eLeg5Ac7Ac). The following structure of the tetrasaccharide repeating unit of the polysac-charide was established: →4)-α-8eLegp5Ac7Ac-(2→6)-α-D-Galp-(1→3)-α-L-FucpNAc-(1→3)-α-D-GlcpNAc-(1→. Functions of the E. coli O108 antigen biosynthetic genes, including seven putative genes for synthesis of 8eLeg5Ac7Ac, were assigned by sequencing the O-antigen gene cluster along with comparison with gene databases and known biosynthetic pathways for related nonulosonic acids.     

Enhanced synthesis of l - threo -3,4-dihydroxyphenylserine by high-density whole-cell biocatalyst of recombinant l -threonine aldolase from Streptomyces avelmitilis

l-threo-3,4-Dihydroxyphenylserine (DOPS) is a chiral unnatural β-hydroxy amino acid used for the treatment of Parkinson disease. We developed a continuous bioconversion system for DOPS production that uses whole-cell biocatalyst of recombinant Escherichia coli expressing l-threonine aldolase (l-TA) genes cloned from Streptomyces avelmitilis MA-4680. Maximum conversion rates were observed at 2 M glycine, 145 mM 3,4-dihydroxybenzaldehyde, 0.75% Triton-X, 5 g E. coli cells/l, pH 6.5 and 10°C. In the optimized condition, overall productivity was 8 g/l, which represents 40 times the synthesis yield possible with no optimization of conditions.     

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.     

Comparative studies on S-adenosyl-l -methionine binding sites of protein N-methyltransferases, using 8-azido-S-adenosyl-l -methionine as photoaffinity probe

Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-l-[methyl-³H]methionine (8-N₃-Ado[methyl-³H]Met), the binding sites for S-adenosyl-l-methionine (AdoMet) of three protein N-methyltransferases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC2.1.1.23); AdoMet:histone-arginin N-methyltransferase (EC2.1.1.23); and AdoMet:cytochromec-lysine N-methyltransferase (EC2.1.1.59)] have been investigated. The incorporation of the photoaffinity label into the enzymes upon UV irradiation was highly specific. In order to define the AdoMet binding sites, the photolabeled enzymes were sequentially digested with trypsin, chymotrypsin, and endoproteinase Glu-C. After each proteolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocratic elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantly different, indicating that their sizes and compositions were different. Amino acid composition analysis of these peptides confirmed further that the AdoMet binding sites of these protein N-methyltransferases are quite different.     

Synonymy and actual affinities of the putative Middle Eocene “New World vulture” Eocathartes Lambrecht , 1935 and “hornbill” Geiseloceros Lambrecht , 1935 (Aves, Ameghinornithidae)

The taxonomy and phylogenetic affinities of the putative “New World vulture”Eocathartes robustus,Lambrecht, 1935 and the “hornbill”Geiseloceros robustus Lambrecht, 1935 from the Middle Eocene of the Geisel Valley in Germany are revised. It is shown that the holotype specimens belong to a single individual, whose osteology closely resembles that ofStrigogyps sapea (Peters, 1987) from the Middle Eocene of Messel (Germany). The species is classified intoStrigogyps robustus (Lambrecht, 1935), n. comb., and provides further evidence for the great similarity between the Eocene avifaunas of the Geisel Valley and Messel.Strigogyps is a representative of the Ameghinornithidae whose phylogenetic affinities are uncertain; there is no fossil record of either Cathartidae (New World vultures) or Bucerotidae (hornbills) from the Geisel Valley.     

Purification and characterization of myo-inositol 6-O-methyltransferase from Vigna umbellata Ohwi et Ohashi

The cyclitol 1d-4-O-methyl-myo-inositol (d-ononitol) is accumulated in certain legumes in response to abiotic stresses. S-Adenosyl-l-methionine:myo-inositol 6-O-methyltransferase (m6OMT), the enzyme which catalyses the synthesis of d-ononitol, was extracted from stems of Vigna umbellata Ohwi et Ohashi and purified to apparent homogeneity by a combination of conventional chromatographic techniques and by affinity chromatography on immobilized S-adenosyl-l-homocysteine (SAH). The purified m6OMT was photoaffinity labelled with S-adenosyl-l-[¹⁴C-methyl]methionine. The native molecular weight was determined to be 106 kDa, with a subunit molecular weight of 40 kDa. Substrate-saturation kinetics of m6OMT for myo-inositol and S-adenosyl-l-methionine (SAM) were Michaelis-Menten type with K _m values of 2.92 mM and 63 M, respectively. The SAH competitively inhibited the enzyme with respect to SAM (K _i of 1.63 M). The enzyme did not require divalent cations for activity, but was strongly inhibited by Mn²⁺, Zn²⁺ and Cu²⁺ and sulfhydryl group inhibitors. The purified m6OMT was found to be highly specific for the 6-hydroxyl group of myo-inositol and showed no activity on other naturally occurring isomeric inositols and inositol O-methyl-ethers. Neither d-ononitol, nor d-3-O-methyl-chiro-inositol, d-1-O-methyl-muco-inositol or d-chiro-inositol (end products of the biosynthetic pathway in which m6OMT catalyses the first step), inhibited the activity of the enzyme.Abbreviations DTT dithiothreitol - m6OMT myo-inositol 6-O-methyltransferase - SAH S-adenosyl-l-homocysteine - SAM S-adenosyl-l-methionine We are greatful to Professor M. Popp (University of Vienna) for helpful discussion and comment. This work was supported by Grant P09595-BIO from the Austrian Science Foundation (FWF).