Substrate specificities of α1,2- and α1,3-galactosyltransferases and characterization of Gmh1p and Otg1p in Schizosaccharomyces pombe.

In the fission yeast Schizosaccharomyces pombe, α1,2- and α1,3-linked D-galactose (Gal) residues are transferred to N- and O-linked oligosaccharides of glycoproteins by galactosyltransferases. Although the galactomannans are important for cell-cell communication in S. pombe (e.g., in nonsexual aggregation), the mechanisms underlying galactosylation in cells remain unclear. Schizosaccharomyces pombe has 10 galactosyltransferase-related genes: seven belonging to glycosyltransferase (GT) family 34 and three belonging GT family 8. Disruption of all 10 α-galactosyltransferases (strain Δ10GalT) has been shown to result in a complete lack of α-Gal residues. Here, we have investigated the function and substrate specificities of galactosyltransferases in S pombe by using strains expressing single α-galactosyltransferases in the Δ10GalT background. High-performance liquid chromatography (HPLC) analysis of pyridylaminated O-linked oligosaccharides showed that two GT family 34 α1,2-galactosyltransferases (Gma12p and Gmh6p) and two GT family 8 α1,3-galactosyltransferases (Otg2p and Otg3p) are involved in galactosylation of O-linked oligosaccharide. Moreover, 1H-NMR of N-glycans revealed that three GT family 34 α1,2-galactosyltransferases (Gmh1p, Gmh2p and Gmh3p) are required for the galactosylation of N-linked oligosaccharides. Furthermore, HPLC and lectin-blot analysis revealed that Otg1p showed α1,3-galactosyltransferase activity under conditions of co-expression with Gmh6p, indicating that α-1,2-linked galactose is required for the galactosylation activity of Otg1p in S. pombe. In conclusion, eight galactosyltransferases have been shown to have activity in S. pombe with different substrate specificities. These findings will be useful for genetically tailoring the galactosylation of both N- and O-glycans in fission yeast.

Stereospecific reduction of the butenolide in strigolactones in plants.

Reductive metabolism of strigolactones (SLs) in several plants was investigated. Analysis of aquaculture filtrates of cowpea and sorghum each fed with four stereoisomers of GR24, the most widely used synthetic SL, revealed stereospecific reduction of the double bond at C-3' and C-4' in the butenolide D-ring with preference for an unnatural 2'S configuration. The cowpea metabolite converted from 2'-epi-GR24 and the sorghum metabolite converted from ent-GR24 had the methyl group at C-4' in the trans configuration with the substituent at C-2', different from the cis configuration of the synthetic H2-GR24 reduced with Pd/C catalyst. The plants also reduced the double bond in the D-ring of 5-deoxystrigol isomers with a similar preference. The metabolites and synthetic H2-GR24 stereoisomers were much less active than were the GR24 stereoisomers in inducing seed germination of the root parasitic weeds Striga hermonthica, Orobanche crenata, and O. minor. These results provide additional evidence of the importance of the D-ring for bioactivity of SLs.

Biosynthesis of anthraquinone derivatives in a Sesamum indicum hairy root culture.

In order to investigate the intermediacy of 2-(4-methylpent-3-en-1-yl)anthraquinone (MPAQ), a possible intermediate for the biosynthesis of anthraquinone derivatives in sesame (Sesamum indicum), 2H-labeled MPAQ was administered to a hairy root culture of S. indicum. Efficient conversion of fed MPAQ to 2-[(Z)-4-methylpenta-1,3-dien-1-yl]anthraquinone ((Z)-MPDEAQ) was observed. Furthermore, administration experiment with 2H-labeled 2-geranyl-1,4-naphthohydroquinone, another possible intermediate, showed that it was converted to MPAQ and (Z)-MPDEAQ. The results clearly demonstrated that these substrates are the actual precursors for the production of (Z)-MPDEAQ. In contrast, neither MPAQ nor 2-geranyl-1,4-naphthohydroquinone was converted to anthrasesamone B and 2,3-epoxyanthrasesamone B, other anthraquinone derivatives in the hairy roots, suggesting that these substrates may not be the common precursors in the biosynthesis of anthraquinone derivatives.

Identification of a characteristic antioxidant, anthrasesamone F, in black sesame seeds and its accumulation at different seed developmental stages.

Assay-guided fractionation of the methanol extract from black seeds of sesame (Sesamum indicum L.) led to the isolation of an active compound that had a 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. This antioxidant was confirmed to be anthrasesamone F, an anthraquinone derivative previously isolated from different black sesame seeds and biogenetically related to other anthrasesamones in sesame roots. The radical scavenging assay showed that anthrasesamone F had more potent activity than Trolox. The content of anthrasesamone F in different parts and at different developmental stages of black sesame seeds was investigated to clarify the accumulation pattern of this antioxidant in the black seeds. Anthrasesamone F was localized in the seed coat of black seeds and accumulated after the seed coat color changed to black. The content of anthrasesamone F increased gradually with seed maturation and drastically on air-drying, the final stage in sesame cultivation.

Dehydroanthrasesamones A and B, anthraquinone derivatives containing a dienyl side-chain from Sesamum indicum hairy roots.

Two additional anthraquinone derivatives containing a (Z)-dienyl side-chain, dehydroanthrasesamones A (2) and B (3), were isolated from a hairy root culture of Sesamum indicum after preventing light throughout all experimental procedures. Their respective structures were determined to be 1-hydroxy-2-[(Z)-4-methylpenta-1,3-dien-1-yl]anthraquinone and 1,4-dihydroxy-2-[(Z)-4-methylpenta-1,3-dien-1-yl]anthraquinone by spectroscopic methods.

Effect of chloride ions on anthrasesamone C production in a Sesamum indicum hairy root culture and identification of the precursor for its abiotic formation.

The content of anthrasesamone C (5), a rare chlorine-containing anthraquinone, in a hairy root culture of Sesamum indicum increased with increasing chloride ion concentration in the culture medium and reached a maximum at 100 mM. However, the amount of anthrasesamone C (5) in the extract obtained from the hairy roots was increased by incubating the extract. This result suggests that anthrasesamone C (5) was produced from an unidentified metabolite by an abiotic process. 2,3-Epoxyanthrasesamone B (1), a precursor for the non-enzymatic formation of anthrasesamone C (5), was isolated from S. indicum hairy roots cultured in a chloride-deficient medium. Its structure was elucidated to be 2,3-epoxy-9,10-dihydroxy-2-(4-methylpent-3-en-1-yl)-2,3-dihydroanthracene-1,4-dione by spectroscopic methods.

Crystal structure of a novel type of ornithine δ-aminotransferase from the hyperthermophilic archaeon Pyrococcus horikoshii.

Ornithine δ-aminotransferase (Orn-AT) activity was detected for the enzyme annotated as a γ-aminobutyrate aminotransferase encoded by PH1423 gene from Pyrococcus horikoshii OT-3. Crystal structures of this novel archaeal ω-aminotransferase were determined for the enzyme in complex with pyridoxal 5'-phosphate (PLP), in complex with PLP and l-ornithine (l-Orn), and in complex with N-(5'-phosphopyridoxyl)-l-glutamate (PLP-l-Glu). Although the sequence identity was relatively low (28%), the main-chain coordinates of P. horikoshii Orn-AT monomer showed notable similarity to those of human Orn-AT. However, the residues recognizing the α-amino group of l-Orn differ between the two enzymes. In human Orn-AT, Tyr55 and Tyr85 recognize the α-amino group, whereas the side chains of Thr92* and Asp93*, which arise from a loop in the neighboring subunit, form hydrogen bonds with the α-amino group of the substrate in P. horikoshii enzyme. Site-directed mutagenesis suggested that Asp93* plays critical roles in maintaining high affinity for the substrate. This study provides new insight into the substrate binding of a novel type of Orn-AT. Moreover, the structure of the enzyme with the reaction-intermediate analogue PLP-l-Glu bound provides the first structural evidence for the "Glu switch" mechanism in the dual substrate specificity of Orn-AT.

Impact of an Olive Leaf Polyphenol 3,4-DHPEA-EDA on Physical Properties of Food Protein Gels.

A cold-water extract of olive leaves (Olea europaea L.) is useful as a texture-improving agent for food protein gels. In this work, the compound contributing to the improvement of gel properties was investigated by using the egg white gel (EWG) as a model for food protein gels. Adding 1.0% (w/v) cold-water extract (OLEx) greatly improved the elasticity (2.1 times), viscosity (4.5 times), and breaking stress (1.4 times) of the EWG. Chemical analyses of the protein revealed that the enhancement of physical properties by OLEx was attributed to protein cross-linking activity of polyphenols. LC/MS and NMR analyses indicated that a major protein cross-linker is the dialdehydic form of demethoxycarbonylelenolic acid linked to hydroxytyrosol (3,4-DHPEA-EDA), which is an aglycone derived from oleuropein, a major polyphenol of olive leaves. These results suggest that 3,4-DHPEA-EDA generated by cold-water extraction from the leaf improves the physical properties, that is, texture, of protein gels.

Retarding activity of 6-O-acyl-D-alloses against plant growth.

The retarding activity of 6-O-dodecanoyl-D-allose against rice growth was higher than that of the octanoate and the decanoate. The activities of 6-O-dodecanoyl-D-glucose, -D-mannose, and -D-galactose against rice seedlings were examined. 6-O-Dodecanoyl-D-allose exhibited the highest activity, suggesting the importance of the alpha-axial hydroxy group at C-3 of D-allose.

Characterization of N- and O-linked galactosylated oligosaccharides from fission yeast species.

The N- and O-linked oligosaccharides from fission yeast Schizosaccharomyces pombe not only contain large amounts of d-mannose (Man) but also contain large amounts of d-galactose (Gal). Although the galactomannans of S. pombe are mainly composed of α1,2- or α1,3-linked Gals, some of the terminal α1,2-linked Gals are found to be linked to pyruvylated ß1,3-linked galactose (PvGal). We have determined the structural characteristics of the N-glycans and O-glycans in three Schizosaccharomyces species (S. japonicus, S. octosporus, and S. cryophilus) using lectin blot, 1H NMR spectroscopy, and size-fractionation high performance liquid chromatography (HPLC), and found that the galactosylation of oligosaccharides was a common feature in fission yeasts. In addition, each of the terminal Galα1,2-, Galß1,3- and non-substituted Man residues exhibited distinct characteristics. A BLAST search of gene databases in Schizosaccharomyces identified genes homologous to pvg1 encoding pyruvyltransferase of S. pombe. These genes, when expressed in an S. pombe pvg1Δ strains, led to the pyruvylation of non-reducing terminal ß-linked Gal, suggesting the biosynthetic pathway of PvGal-containing oligosaccharides is highly conserved in fission yeasts.

Biosynthetic origin of 2,3-epoxysesamone in a Sesamum indicum hairy root culture.

The incorporation of [1-(13)C]glucose into 2,3-epoxysesamone, the main prenylnaphthoquinone in a hairy root culture of Sesamum indicum, indicated that the naphthoquinone moiety and dimethylallyl group were respectively derived from o-succinylbenzoate produced through a shikimate pathway and non-mevalonate pathway. The labeling pattern also demonstrated that prenylation occurred at C-2 in 1,4-dihydroxy-2-naphthoate.

Isolation and photoisomerization of a new anthraquinone from hairy root cultures of Sesamum indicum.

An unstable anthraquinone was isolated from hairy root cultures of Sesamum indicum after preventing light throughout all experimental procedures. The structure of the (Z)-isomer of a previously isolated anthraquinone was determined to be 2-[(Z)-4-methylpenta-1,3-dien-1-yl]anthraquinone by spectroscopic methods. This compound was readily isomerized to the known (E)-isomer under light.

Production and application of a rare disaccharide using sucrose phosphorylase from Leuconostoc mesenteroides.

Sucrose phosphorylase (SPase) from Leuconostoc mesenteroides exhibited activity towards eight ketohexoses, which behaved as D-glucosyl acceptors, and α-D-glucose-1-phosphate (G1P), which behaved as a donor. All eight of these ketohexoses were subsequently transformed into the corresponding d-glucosyl-ketohexoses. Of the eight ketohexoses evaluated in the current study, d-allulose behaved as the best substrate for SPase, and the resulting d-glucosyl-d-alluloside product was found to be a non-reducing sugar with a specific optical rotation of [α]D(20) + 74.36°. D-Glucosyl-D-alluloside was identified as α-D-glucopyranosyl-(1→2)-ß-D-allulofuranoside by NMR analysis. D-Glucosyl-D-alluloside exhibited an inhibitory activity towards an invertase from yeast with a Km value of 50 mM, where it behaved as a competitive inhibitor with a Ki value of 9.2 mM. D-Glucosyl-D-alluloside was also successfully produced from sucrose using SPase and D-tagatose 3-epimerase. This process also allowed for the production of G1P from sucrose and d-allulose from D-fructose, which suggested that this method could be used to prepare d-glucosyl-d-alluloside without the need for expensive reagents such as G1P and d-allulose.

Anthrasesamones from roots of Sesamum indicum.

Three anthraquinones, named anthrasesamones A, B and C, were isolated from the roots of Sesamum indicum, and their respective structures were determined to be 1-hydroxy-2-(4-methylpent-3-enyl)anthraquinone, 1,4-dihydroxy-2-(4-methylpent-3-enyl)anthraquinone and 2-chloro-1,4-dihydroxy-3-(4-methylpent-3-enyl)anthraquinone on the basis of spectroscopic evidence. Two known anthraquinones were also isolated for the first time from S. indicum roots and characterized as 2-(4-methylpent-3-enyl)anthraquinone and (E)-2-(4-methylpenta-1,3-dienyl)anthraquinone. Anthrasesamone C is a rare chlorinated anthraquinone in higher plants.

Heliolactone, a non-sesquiterpene lactone germination stimulant for root parasitic weeds from sunflower.

Root exudates of sunflower (Helianthus annuus L.) line 2607A induced germination of seeds of root parasitic weeds Striga hermonthica, Orobanche cumana, Orobanche minor, Orobanche crenata, and Phelipanche aegyptiaca. Bioassay-guided purification led to the isolation of a germination stimulant designated as heliolactone. FT-MS analysis indicated a molecular formula of C20H24O6. Detailed NMR spectroscopic studies established a methylfuranone group, a common structural component of strigolactones connected to a methyl ester of a C14 carboxylic acid via an enol ether bridge. The cyclohexenone ring is identical to that of 3-oxo-α-ionol and the other part of the molecule corresponds to an oxidized carlactone at C-19. It is a carlactone-type molecule and functions as a germination stimulant for seeds of root parasitic weeds. Heliolactone induced seed germination of the above mentioned root parasitic weeds, while dehydrocostus lactone and costunolide, sesquiterpene lactones isolated from sunflower root exudates, were effective only on O. cumana and O. minor. Heliolactone production in aquacultures increased when sunflower seedlings were grown hydroponically in tap water and decreased on supplementation of the culture with either phosphorus or nitrogen. Costunolide, on the other hand, was detected at a higher concentration in well-nourished medium as opposed to nutrient-deficient media, thus suggesting a contrasting contribution of heliolactone and the sesquiterpene lactone to the germination of O. cumana under different soil fertility levels.

Biosynthetic origin of 2-geranyl-1,4-naphthoquinone and its related anthraquinone in a Sesamum indicum hairy root culture.

In order to clarify the biosynthetic origin of 2-geranyl-1,4-naphthoquinone and its biogenetically related anthraquinone, which are possible intermediates of anthrasesamones, [1-(13)C]glucose was administered to a hairy root culture of Sesamum indicum. The labeling patterns of these quinone derivatives indicated that the naphthoquinone ring and geranyl side-chain of geranylnaphthoquinone were respectively biosynthesized through the shikimate and methylerythritol phosphate pathways, and that these quinone derivatives have the same biosynthetic origin.

2-Geranyl-1,4-naphthoquinone, a possible intermediate of anthraquinones in a Sesamum indicum hairy root culture.

2-Geranyl-1,4-naphthoquinone was isolated from the hairy root culture of Sesamum indicum. The structure was determined to be 2-[(E)-3,7-dimethylocta-2,6-dienyl]-1,4-naphthoquinone on the basis of spectroscopic evidence and chemical synthesis. The production of anthrasesamones A, B and C by the hairy root culture was also confirmed for the first time.

Anthrasesamones D and E from Sesamum indicum roots.

Two anthraquinone derivatives, named anthrasesamones D and E, were isolated from the roots of Sesamum indicum. Their respective structures were determined to be 1,2,4-trihydroxy-3-(4-methylpent-3-enyl)anthraquinone and 1,2-dihydroxy-3-(4-methylpent-3-enyl)anthraquinone on the basis of spectroscopic evidence.

Absorption and metabolism of bisphenol A, a possible endocrine disruptor, in the aquatic edible plant, water convolvulus (Ipomoea aquatica).

Water convolvulus, a vegetable, absorbed bisphenol A (BPA), an endocrine disruptor, from the medium. One week later, no BPA could be detected in the plant, indicating that BPA had been metabolized in the plant. BPA monoglucoside was detected as the BPA base at ca. 10% in the roots, some in the stems, but none in the leaves. (2)H-NMR analyses of MeOH extracts and hydrolyzates of the plant treated with BPA-d(16) showed the presence of metabolites (ca. 7% and 26%, respectively, as BPA equivalents) other than the glucoside. Over 50% of BPA might be polymerized and/or tightly bound in the plant residues.

Possible antitumor promoters in Spinacia oleracea (spinach) and comparison of their contents among cultivars.

Spinach leaves were found to contain two potent antitumor promoters as detected by the activity against tumor promoter-induced Epstein-Barr virus activation. The active components were identified as 1-O-alpha-linolenoyl-2-O-(7Z,10Z,13Z)-hexadecatrienoyl-3-O-beta-D-galactopyranosyl-sn-glycerol and 1,2-di-O-alpha-linolenoyl-3-O-beta-D-galactopyranosyl-sn-glycerol by spectroscopic data and some chemical and enzymatic reactions. Their contents significantly varied with the cultivar and with the culture conditions.