Discovery of Anomer-Inverting Transglycosylase: Cyclic Glucohexadecaose-Producing Enzyme from Xanthomonas, a Phytopathogen.

Various Xanthomonas species cause well-known plant diseases. Among various pathogenic factors, the role of α-1,6-cyclized ß-1,2-glucohexadecaose (CßG16α) produced by Xanthomonas campestris pv. campestris was previously shown to be vital for infecting model organisms, Arabidopsis thaliana and Nicotiana benthamiana. However, enzymes responsible for biosynthesizing CßG16α are essentially unknown, which limits the generation of agrichemicals that inhibit CßG16α synthesis. In this study, we discovered that OpgD from X. campestris pv. campestris converts linear ß-1,2-glucan to CßG16α. Structural and functional analyses revealed OpgD from X. campestris pv. campestris possesses an anomer-inverting transglycosylation mechanism, which is unprecedented among glycoside hydrolase family enzymes.

Organic Synthesis of New Secosteroids from Fucosterol, Its Intestinal Absorption by Caco-2 Cells, and Simulation of the Biological Activities of Vitamin D.

We previously examined the cellular uptake of six types of vitamin D in human intestinal Caco-2 cells. Since vitamins D5-D7 were commercially unavailable, we synthesized these compounds organically before studying them. This process led us to understand that new secosteroids could be generated as vitamin D candidates, depending on the sterol used as the starting material. We obtained two new secosteroids-compounds 3 and 4-from fucosterol in the current study. We investigated the intestinal absorption of these compounds using Caco-2 cells cultured in Transwells and compared the results with vitamin D3, a representative secosteroid. The intestinal absorption of compound 4 was comparable to that of vitamin D3. Compound 3 showed similar uptake levels but transported about half as much as vitamin D3. These compounds demonstrated intestinal absorption at the cellular level. Vitamin D is known for its diverse biological activities manifest after intestinal absorption. Using PASS online simulation, we estimated the biological activity of compound 3's activated form. In several items indicated by PASS, compound 3 exhibited stronger biological activity than vitamins D2-D7 and was also predicted to have unique biological activities.

Molecular mechanism for endo-type action of glycoside hydrolase family 55 endo-ß-1,3-glucanase on ß1-3/1-6-glucan.

The glycoside hydrolase family 55 (GH55) includes inverting exo-ß-1,3-glucosidases and endo-ß-1,3-glucanases, acting on laminarin, which is a ß1-3/1-6-glucan consisting of a ß1-3/1-6-linked main chain and ß1-6-linked branches. Despite their different modes of action toward laminarin, endo-ß-1,3-glucanases share with exo-ß-1,3-glucosidases conserved residues that form the dead-end structure of subsite -1. Here, we investigated the mechanism of endo-type action on laminarin by GH55 endo-ß-1,3-glucanase MnLam55A, identified from Microdochium nivale. MnLam55A, like other endo-ß-1,3-glucanases, degraded internal ß-d-glucosidic linkages of laminarin, producing more reducing sugars than the sum of d-glucose and gentiooligosaccharides detected. ß1-3-Glucans lacking ß1-6-linkages in the main chain were not hydrolyzed. NMR analysis of the initial degradation of laminarin revealed that MnLam55A preferentially cleaved the nonreducing terminal ß1-3-linkage of the laminarioligosaccharide moiety at the reducing end side of the main chain ß1-6-linkage. MnLam55A liberates d-glucose from laminaritriose and longer laminarioligosaccharides, but kcat/Km values to laminarioligosaccharides (≤4.21 s-1 mM-1) were much lower than to laminarin (5920 s-1 mM-1). These results indicate that ß-glucan binding to the minus subsites of MnLam55A, including exclusive binding of the gentiobiosyl moiety to subsites -1 and -2, is required for high hydrolytic activity. A crystal structure of MnLam55A, determined at 2.4 Å resolution, showed that MnLam55A adopts an overall structure and catalytic site similar to those of exo-ß-1,3-glucosidases. However, MnLam55A possesses an extended substrate-binding cleft that is expected to form the minus subsites. Sequence comparison suggested that other endo-type enzymes share the extended cleft. The specific hydrolysis of internal linkages in laminarin is presumably common to GH55 endo-ß-1,3-glucanases.

Chemical synthesis of oligosaccharide derivatives with partial structure of ß1-3/1-6 glucan, using monomeric units for the formation of ß1-3 and ß1-6 glucosidic linkages.

ß1-3/1-6 Glucans, known for their diverse structures, comprise a ß1-3-linked main chain and ß1-6-linked short branches. Laminarin, a ß1-3/1-6 glucan extracted from brown seaweed, for instance, includes ß1-6 linkages even in the main chain. The diverse structures provide various beneficial functions for the glucan. To investigate the relationship between structure and functionality, and to enable the characterization of ß1-3/1-6 glucan-metabolizing enzymes, oligosaccharides containing the exact structures of ß1-3/1-6 glucans are required. We synthesized the monomeric units for the synthesis of ß1-3/1-6 mixed-linked glucooligosaccharides. 2-(Trimethylsilyl)ethyl 2-O-benzoyl-4,6-O-benzylidene-ß-d-glucopyranoside served as an acceptor in the formation of ß1-3 linkages. Phenyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(tert-butyldiphenylsilyl)-1-thio-ß-d-glucopyranoside and phenyl 2,3-di-O-benzoyl-4,6-di-O-levulinyl-1-thio-ß-d-glucopyranoside acted as donors, synthesizing acceptors suitable for the formation of ß1-3- and ß1-6-linkages, respectively. These were used to synthesize a derivative of Glcß1-6Glcß1-3Glcß1-3Glc, demonstrating that the proposed route can be applied to synthesize the main chain of ß-glucan, with the inclusion of both ß1-3 and ß1-6 linkages.

High-temperature solvent-free synthesis of low-molecular-weight organogelators consisting of starch-derived 1,5-anhydro-d-glucitol coupled with fatty acids.

We previously developed low-molecular-weight organogelators that can gel various types of oils by introducing various fatty acids into 1,5-anhydro-d-glucitol (1,5-AG), a cyclic polyol derived from starch. These gelators are composed of ester bonds between naturally occurring materials. We have established a method to synthesize these gelators by heating powdered 1,5-AG and powdered fatty acids to 230 °C to liquefy the compounds without using organic solvents or activated fatty acids and then performing the esterification reaction without a catalyst or with a base catalyst. This has created the possibility of synthesizing these gelling agents at very low cost.

Development of Low-Molecular-Mass Organogelators: Synthesis and Physical Properties of N-Linear Saturated Fatty Acyl-GABAs and Their Ester Derivatives.

We determined that compounds in which γ-aminobutyric acid (GABA) and linear saturated fatty acids of various lengths are amide-bonded, as found in the human brain, have the ability to gelate organic solvents. We also synthesized compounds of these GABA derivatives attached to 1,5-anhydro-d-glucitol (1,5-AG) or d-glucopyranose (Glc) via ester linkages, and these compounds were also found to be able to gelate organic solvents. From the comparative experiments of gelation using various lengths of N-linear saturated fatty acyl-GABAs and their ester derivatives, it was determined that the compound of N-tetradecanoic acyl-GABA bonded to 1,5-AG via ester linkage (C14GABA-AG) had a particularly high gel hardness and could gelate various organic solvents. Furthermore, field-emission scanning electron microscopy observations revealed the formation of a fibrous structure, which encapsulates the organic solvent and forms a gel. A variable-temperature Fourier transform infrared spectroscopy analysis revealed that the alkyl chains of N-linear saturated fatty acyl-GABAs are packed with an all-trans conformation, whereas the alkyl chains of the ester compounds attached to 1,5-AG or Glc are slightly skewed from the all-trans conformation due to the intermolecular hydrogen bonding of the amide groups. Here, we report the synthesis and analysis of N-linear saturated fatty acyl-GABA derivatives and the gelation mechanism.

Uptake of Vitamins D2, D3, D4, D5, D6, and D7 Solubilized in Mixed Micelles by Human Intestinal Cells, Caco-2, an Enhancing Effect of Lysophosphatidylcholine on the Cellular Uptake, and Estimation of Vitamins D' Biological Activities.

Vitamins D have various biological activities, as well as intestinal calcium absorption. There has been recent concern about insufficient vitamin D intake. In addition to vitamins D2 and D3, there are lesser-known vitamins D4-D7. We synthesized vitamins D5-D7, which are not commercially available, and then evaluated and compared the mixed micelles-solubilized vitamins D uptake by Caco-2 cells. Except for vitamin D5, the uptake amounts of vitamins D4-D7 by differentiated Caco-2 cells were similar to those of vitamins D2 and D3. The facilitative diffusion rate in the ezetimibe inhibited pathway was approximately 20% for each vitamin D type, suggesting that they would pass through the pathway at a similar rate. Lysophosphatidylcholine enhanced each vitamin D uptake by approximately 2.5-fold. Lysophosphatidylcholine showed an enhancing effect on vitamin D uptake by reducing the intercellular barrier formation of Caco-2 cells by reducing cellular cholesterol, suggesting that increasing the uptakes of vitamins D and/or co-ingesting them with lysophosphatidylcholine, would improve vitamin D insufficiency. The various biological activities in the activated form of vitamins D4-D7 were estimated by Prediction of Activity Spectra for Substances (PASS) online simulation. These may have some biological activities, supporting the potential as nutritional components.

Supramolecular fibrous gels with helical pitch tunable by polarity of alcohol solvents.

A sugar-based, low-molecular-weight gelator 16AG, can gelatinize primary alcohols by forming supramolecular fibers. We obtained non-helical, tape-like fibers in methanol and ethanol but helical fibers in alcohols with at least three carbons. The pitch of the helical fibers became shorter with increasing carbon number.

Supramolecular Organogelation Directed by Weak Noncovalent Interactions in Palmitoylated 1,5-Anhydro-d-Glucitol Derivatives.

We synthesized a series of novel alicyclic compounds by modifying 1,5-anhydro-d-glucitol with two to four palmitoyl chains, and we explored their self-assembly and gelation behaviors in paraffin. The obtained organogels were studied by field emission scanning electron microscopy, atomic force microscopy, variable-temperature Fourier transform IR spectroscopy, X-ray diffraction analysis, polarized optical microscopy, and transmission spectroscopy. While all the palmitoylated derivatives spontaneously formed fibrous networks and gelated the paraffin, an acetylated derivative of 1,5-anhydro-d-glucitol did not gelatinize the solvent, thus indicating the importance of aliphatic chains for gelation. Interestingly, α- and ß- d-glucopyranose with five palmitoyl chains neither gelatinized the solvent nor formed fibrous networks, thus suggesting that the absence of C-1 substitution in 1,5-anhydro-d-glucitol is important for gelation. Fourier transform IR spectroscopy suggested that the formation of weak hydrogen bonds between the carbonyl groups and the C-H groups was the driving force for formation of the supramolecular fibers and for gelation of the solvent.

Diversity in γ-oryzanol profiles of Japanese black-purple rice varieties.

The γ-oryzanol contents and the composition of steryl ferulates distributed in Japanese pigmented rice varieties were investigated using the high-performance liquid chromatography-ultraviolet detection method for the purpose of expanding their utilisation. The average γ-oryzanol content in nine black-purple, four red, four green and three brown rice varieties was 54.2, 47.3, 44.3 and 43.3 mg γ-oryzanol equivalent/100 g dried weight, respectively. Among the nine varieties of black-purple rice, five varieties showed steryl ferulate composition similar to that of brown, red and green varieties. In contrast, the composition of steryl ferulates in other four black-purple rice varieties was partially specific and was characterised by a low amount of campesteryl ferulate and high of campestanyl ferulate and stigmastanyl ferulate. The latter two steryl ferulates have been recognised as minor components of γ-oryzanol in rice and as major components in wheat and corn. These results indicate that the compositions of steryl ferulates vary among Japanese black-purple rice varieties.

Simultaneous Synthesis of Vitamins D2, D4, D5, D6, and D7 from Commercially Available Phytosterol, ß-Sitosterol, and Identification of Each Vitamin D by HSQC NMR.

We succeeded in simultaneously synthesizing the vitamin D family, vitamins D2, D4, D5, D6, and D7, from ß-sitosterol, which is sold as a commercially available reagent from Tokyo Chemical Industry Co., Ltd. It is officially sold as a mixture of four phytosterols {ß-sitosterol (40-45%), campesterol (20-30%), stigmasterol, and brassicasterol}. Owing to this, we anticipated that, using this reagent, various vitamin D analogs could be synthesized simultaneously. We also synthesized vitamin D3 from pure cholesterol and analyzed and compared all vitamin D analogs (D2, D3, D4, D5, D6, and D7) by HSQC NMR. We succeeded in clearly demonstrating the difference in the NMR chemical shifts for each vitamin D analog.

Identification, characterization, and structural analyses of a fungal endo-ß-1,2-glucanase reveal a new glycoside hydrolase family.

endo-ß-1,2-Glucanase (SGL) is an enzyme that hydrolyzes ß-1,2-glucans, which play important physiological roles in some bacteria as a cyclic form. To date, no eukaryotic SGL has been identified. We purified an SGL from Talaromyces funiculosus (TfSGL), a soil fungus, to homogeneity and then cloned the complementary DNA encoding the enzyme. TfSGL shows no significant sequence similarity to any known glycoside hydrolase (GH) families, but shows significant similarity to certain eukaryotic proteins with unknown functions. The recombinant TfSGL (TfSGLr) specifically hydrolyzed linear and cyclic ß-1,2-glucans to sophorose (Glc-ß-1,2-Glc) as a main product. TfSGLr hydrolyzed reducing-end-modified ß-1,2-gluco-oligosaccharides to release a sophoroside with the modified moiety. These results indicate that TfSGL is an endo-type enzyme that preferably releases sophorose from the reducing end of substrates. Stereochemical analysis demonstrated that TfSGL is an inverting enzyme. The overall structure of TfSGLr includes an (α/α)6 toroid fold. The substrate-binding mode was revealed by the structure of a Michaelis complex of an inactive TfSGLr mutant with a ß-1,2-glucoheptasaccharide. Mutational analysis and action pattern analysis of ß-1,2-gluco-oligosaccharide derivatives revealed an unprecedented catalytic mechanism for substrate hydrolysis. Glu-262 (general acid) indirectly protonates the anomeric oxygen at subsite -1 via the 3-hydroxy group of the Glc moiety at subsite +2, and Asp-446 (general base) activates the nucleophilic water via another water. TfSGLr is apparently different from a GH144 SGL in the reaction and substrate recognition mechanism based on structural comparison. Overall, we propose that TfSGL and closely-related enzymes can be classified into a new family, GH162.

Development of Novel Low-Molecular-Mass Oil-gelling Agents: Synthesis and Physical Properties of 1,5-Anhydro-D-glucitol and 1,5-Anhydro-D-mannitol Protected with Saturated Linear Fatty Acids.

We have developed a novel low-molecular-mass oil-gelling agent that is electrically neutral, has no nitrogen atoms and consists only of cyclic sugar alcohols and saturated linear fatty acids. The cyclic sugar alcohols were 1,5-anhydro-D-glucitol (1,5-AG) and 1,5-anhydro-D-mannitol (1,5-AM) derived from starch via 1,5-anhydro-D-fructose. Various saturated linear fatty acids with 10 to 18 and 22 carbon atoms were introduced into all the hydroxy groups of 1,5-AG. Various saturated linear fatty acids with 13 to 18 and 22 carbon atoms were introduced into all the hydroxy groups of 1,5-AM. Initially, the gelling ability increased as the carbon number increased, but the gelling ability decreased as the carbon number increased beyond 17 carbons. This trend was similar for both 1,5-AG and 1,5-AM. A comparison of 1,5-AG and 1,5-AM derivatives revealed that 1,5-AG derivatives had greater gelling abilities for different kinds of oils at the same fatty acid length. Further, it was confirmed by SEM observations that a three-dimensional fibrous structure was formed, and this network structure formed the gel and held the oil. Here, we report the synthesis and characteristics of a novel low-molecular-weight gelling agent and its gelation mechanism.

Degradation of Fucoxanthin to Elucidate the Relationship between the Fucoxanthin Molecular Structure and Its Antiproliferative Effect on Caco-2 Cells.

Fucoxanthin has an antiproliferative effect on cancer cells, but its detailed structure⁻activity correlation has not yet been elucidated. To elucidate this correlation, fucoxanthin was degraded by ozonolysis. The degraded compounds of fucoxanthin obtained by ozonolysis were purified by HPLC and analyzed by NMR. The polyene chain of fucoxanthin was cleaved by ozonolysis, and the fucoxanthin was divided into two types of cyclohexyl derivatives, one with a ß,γ-epoxy ketone group and the other with an allenic bond. In order to elucidate the structure⁻activity correlation, Caco-2 cells (human colorectal carcinoma) were treated with fucoxanthin degradation compounds. It was found that the entire structure of fucoxanthin is not essential for its antiproliferative effect and that even a partial structure exerts this effect.

Synthesis of three deoxy-sophorose derivatives for evaluating the requirement of hydroxy groups at position 3 and/or 3' of sophorose by 1,2-ß-oligoglucan phosphorylases.

Sophorose (Sop2) is known as a powerful inducer of cellulases in Trichoderma reesei, and in recent years 1,2-ß-D-oligoglucan phosphorylase (SOGP) has been found to use Sop2 in synthetic reactions. From the structure of the complex of SOGP with Sop2, it was predicted that both the 3-hydroxy group at the reducing end glucose moiety of Sop2 and the 3'-hydroxy group at the non-reducing end glucose moiety of Sop2 were important for substrate recognition. In this study, three kinds of 3- and/or 3'-deoxy-Sop2 derivatives were synthesized to evaluate this mechanism. The deoxygenation of the 3-hydroxy group of D-glucopyranose derivative was performed by radical reduction using a toluoyl group as a leaving group. The utilization of a toluoyl group that plays two roles (a leaving group for the deoxygenation and a protecting group for a hydroxy group) resulted in efficient syntheses of the three target compounds. The NMR spectra of the two final compounds (3-deoxy- and 3,3'-dideoxy-Sop2) suggested that the glucose moiety of the reducing end of Sop2 can easily take on a furanose structure (five-membered ring structure) by deoxygenation of the 3-hydroxy group of Sop2. In addition, the ratio of the five- and six-membered ring structures changed depending on the temperature. The SOGPs exhibited remarkably lower specific activity for 3'-deoxy- and 3,3'-dideoxy-Sop2, indicating that the 3'-hydroxy group of Sop2 is important for substrate recognition by SOGPs.

The content and distribution of steryl ferulates in wheat produced in Japan.

Oryzanol contained in rice bran is a complex mixture of steryl ferulates (SFs) with many identified health benefits. Recently, SF has been shown to exist in other cereals such as wheat, rye, and corn. In this study, SFs in several wheats produced in Japan were analyzed. For instance, SF content of whole wheat grain, Yumekaori (Japan) was 15.2 ± 1.4 mg-oryzanol-equivalent/100 g grain, while that of the imported one, 1CW (Canada) was 11.4 ± 1.3 mg-oryzanol-equivalent/100 g grain. The main SF components in the examined wheats were campesteryl ferulate, campestanyl ferulate, and sitostanyl ferulate. SF distribution in whole wheat grain was investigated using 14 fractions produced by a conventional test milling machine. SF was intensively accumulated in the four bran fractions (24 - 95 mg-oryzanol-equivalent/100 g bran fraction). These results suggest that the wheat bran would be an important source of SF.

Enzymatic Synthesis of 1,5-Anhydro-4-O-ß-D-glucopyranosyl-D-fructose Using Cellobiose Phosphorylase and Its Spontaneous Decomposition via ß-Elimination.

Cellobiose phosphorylase from Cellvibrio gilvus was used to prepare 1,5-anhydro-4-O-ß-D-glucopyranosyl-D-fructose [ßGlc(1→4)AF] from 1,5-anhydro-D-fructose and α-D-glucose 1-phosphate. ßGlc(1→4)AF decomposed into D-glucose and ascopyrone T via ß-elimination. Higher pH and temperature caused faster decomposition. However, decomposition proceeded significantly even under mild conditions. For instance, the half-life of ßGlc(1→4)AF was 17 h at 30 °C and pH 7.0. Because ßGlc(1→4)AF is a mimic of cellulose, in which the C2 hydroxyl group is oxidized, such decomposition may occur in oxidized cellulose in nature. Here we propose a possible oxidizing pathway by which this occurs.

Screening, expression, and characterization of an anti-human oxidized low-density lipoprotein single-chain variable fragment.

Increased levels of oxidized low-density lipoprotein (OxLDL) in the blood circulation are correlated with atherosclerosis. Monoclonal antibody-based detection systems have been reported for OxLDL. We identified novel single-chain variable fragments (scFvs) having affinity for human OxLDL and related ligands. We constructed an scFv library from nonimmunized human spleen mRNA. Two types (γ+κ and µ+λ) of scFv phage libraries were enriched by biopanning, and five scFv clones with affinity for OxLDL were identified. The γκ5 scFv, which showed the highest affinity for OxLDL, was cloned into pET-22b(+) and expressed in Escherichia coli BL21(DE3). γκ5, expressed as an inclusion body in BL21(DE3), was refolded and purified. The specificity and sensitivity of γκ5 were analyzed using enzyme-linked immunosorbent assays (ELISAs). The γκ5 scFv showed affinity for OxLDL and acetylated LDL. The sensitivity of γκ5 to low concentrations (1-2 µg/mL) of OxLDL was higher than that to AcLDL and LDL. Finally, we developed a sandwich ELISA using γκ5 and CTLD14 (a lectin-like OxLDL receptor-1 ligand recognition region), which allowed specific detection of OxLDL at a level below 0.1 µg/mL. Our results indicated that the γκ5 scFv was a promising molecule for the detection of modified LDL at very low concentrations.

Fucoxanthin Derivatives: Synthesis and their Chemical Properties.

Novel fucoxanthin derivatives that could change the size of mixed micelles were synthesized. The mixed micelles under consideration consist of a bile acid and some additives. To change the affinity against a bile acid, we designed the synthesis of a fucoxanthin-lithocholic acid complex. Lithocholic acid is one of the bile acids. The 3-OH on lithocholic acid was protected by a levulinyl group, and the protected lithocholic acid was selectively coupled via an ester linkage to the 3-OH on fucoxanthin to obtain levulinyl-protected lithocholyl fucoxanthin (LevLF). The levulinyl group was then selectively deprotected using hydrazine to obtain a lithocholyl fucoxanthin (LF). The average sizes of the micelles that contained these compounds (fucoxanthin, LevLF, and LF) with a bile acid (sodium taurocholate) were measured. The LevLF induced larger micelles than fucoxanthin or LF. Interestingly, the addition of 1-oleoyl-rac-glycerol induced a more efficient change in the micelle size. The large micelles grew larger, and the small micelles became smaller. Triple-mixed micelles with LevLF, sodium taurocholate, and 1-oleoyl-rac-glycerol formed the largest micelle with a diameter of 68 nm. On the other hand, triple-mixed micelles using LF, sodium taurocholate, and 1-oleoyl-rac-glycerol made the smallest micelles with diameters as low as 12 nm. We also investigated the hydrolysis of these compounds with enzymes (esterase from porcine liver, lipase from porcine pancreas, and cholesterol esterase from Pseudomonas sp.). The ester linkage between the lithocholic acid and fucoxanthin of LevLF was hydrolyzed with cholesterol esterase. In addition, the intestinal absorption was examined with Caco-2 cells, and no advantageous change in absorption efficiency was observed by chemically modifying the fucoxanthin unless different micelles sizes and increasing hydrophobicity are induced.

Lectin-like oxidized LDL receptor-1 is palmitoylated and internalizes ligands via caveolae/raft-dependent endocytosis.

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is an endothelial scavenger receptor that is important for oxidized low-density lipoprotein uptake. LOX-1 functions as an oligomer; however, little is known about the oligomeric complex and ligand processing after recognition by LOX-1. Here, we found that LOX-1 recognized and internalized ligands through the caveolae/raft-dependent endocytosis pathway in human coronary artery endothelial cells. Furthermore, we demonstrated that LOX-1 was palmitoylated and that both cysteine 36 and cysteine 46 were necessary for the recruitment of LOX-1 into raft microdomains and for its ligand uptake ability.