Yasushi Mitsuishi

Suppression and acceleration of cell elongation by integration of xyloglucans in pea stem segments.

Xyloglucan is a key polymer in the walls of growing plant cells. Using split pea stem segments and stem segments from which the epidermis had been peeled off, we demonstrate that the integration of xyloglucan mediated by the action of wall-bound xyloglucan endotransglycosylase suppressed cell elongation, whereas that of its fragment oligosaccharide accelerated it. Whole xyloglucan was incorporated into the cell wall and induced the rearrangement of cortical microtubules from transverse to longitudinal; in contrast, the oligosaccharide solubilized xyloglucan from the cell wall and maintained the microtubules in a transverse orientation. This paper proposes that xyloglucan metabolism controls the elongation of plant cells.

Purification, Characterization, Cloning, and Expression of a Novel Xyloglucan-specific Glycosidase, Oligoxyloglucan Reducing End-specific Cellobiohydrolase

A novel oligoxyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase (OXG-RCBH), with a molecular mass of 97 kDa and a pI of 6.1, was isolated from the fungus Geotrichum sp. M128. Analysis of substrate specificity using various xyloglucan oligosaccharide structures revealed that OXG-RCBH had exoglucanase activity. It recognized the reducing end of oligoxyloglucan and released two glucosyl residue segments from the main chain. The full-length cDNA encoding OXG-RCBH was cloned and sequenced, and it had a 2436-bp open reading frame encoding an 812amino acid protein. The deduced protein showed ∼35% identity to members of glycoside hydrolase family 74. The cDNA encoding OXG-RCBH was then expressed inEscherichia coli. Although the recombinant protein was expressed as an inclusion body, renaturation was successful, and enzymatically active recombinant OXG-RCBH was obtained.

Purification, characterization, cDNA cloning, and expression of a xyloglucan endoglucanase from Geotrichum sp. M1281

A novel xyloglucan‐specific endo‐β‐1,4‐glucanase (XEG), xyloglucanase, with a molecular mass of 80 kDa and a pI of 4.8, was isolated from the fungus Geotrichum sp. M128. It was found to be an endoglucanase active toward xyloglucan and not active toward carboxymethylcellulose, Avicel, or barley 1,3‐1,4‐β‐glucan. Analysis of the precise substrate specificity using various xyloglucan oligosaccharide structures revealed that XEG has at least four subsites (−2 to +2) and specifically recognizes xylose branching at the +1 and +2 sites. The full‐length cDNA encoding XEG was cloned and sequenced. It consists of a 2436‐bp open reading frame encoding a 776‐amino acid protein. From its deduced amino acid sequence, XEG can be classified as a family 74 glycosyl hydrolase. The cDNA encoding XEG was then expressed in Escherichia coli, and enzymatically active recombinant XEG was obtained.

Cloning and Characterization of Two Xyloglucanases from Paenibacillus sp. Strain KM21

ABSTRACT Two xyloglucan-specific endo-β-1,4-glucanases (xyloglucanases [XEGs]), XEG5 and XEG74, with molecular masses of 40 kDa and 105 kDa, respectively, were isolated from the gram-positive bacterium Paenibacillus sp. strain KM21, which degrades tamarind seed xyloglucan. The genes encoding these XEGs were cloned and sequenced. Based on their amino acid sequences, the catalytic domains of XEG5 and XEG74 were classified in the glycoside hydrolase families 5 and 74, respectively. XEG5 is the first xyloglucanase belonging to glycoside hydrolase family 5. XEG5 lacks a carbohydrate-binding module, while XEG74 has an X2 module and a family 3 type carbohydrate-binding module at its C terminus. The two XEGs were expressed in Escherichia coli, and recombinant forms of the enzymes were purified and characterized. Both XEGs had endoglucanase active only toward xyloglucan and not toward Avicel, carboxymethylcellulose, barley β-1,3/1,4-glucan, or xylan. XEG5 is a typical endo-type enzyme that randomly cleaves the xyloglucan main chain, while XEG74 has dual endo- and exo-mode activities or processive endo-mode activity. XEG5 digested the xyloglucan oligosaccharide XXXGXXXG to produce XXXG, whereas XEG74 digestion of XXXGXXXG resulted in XXX, XXXG, and GXXXG, suggesting that this enzyme cleaves the glycosidic bond of unbranched Glc residues. Analyses using various oligosaccharide structures revealed that unique structures of xyloglucan oligosaccharides can be prepared with XEG74.

Determination of structural isomers of xyloglucan octasaccharides using post-source decay fragment analysis in MALDI-TOF mass spectrometry

Abstract Post-source decay (PSD) fragment analyses by MALDI-TOFMS were applied to the characterization of two analogous structure isomers of xyloglucan octasaccharides (XXLGol and XLXGol) from Tamarind seed. Almost all possible fragment ions were detected by a mutli-site cleavage at the glycosidic linkages. The same fragment ions were observed in their spectra, but the relative intensities of some of the ions differed greatly. The detailed investigation of the relative intensities of the fragment ions enabled definitely to distinguish the analogous structure isomers of the highly branched octasaccharides.

Site-directed mutagenesis of the putative catalytic residues of Trichoderma reesci cellobiohydrolase I and endoglucanase I

Site directed mutagenesis has been performed to test hypotheses concerning the putative active sites of Trichoderma reesci cellobiohydrolase I and endoglucanase I. It is shown that mutagenesis of the residue 1:126, previously proposed to be the proton donor in CBHI, did not totally inactive the enzyme while mutagenesis of the residue 1:127 in the homologous enzyme EG1 resulted in complete loss of activity. These results are compared with those obtained in similar studies of other glucanases and the effects on enzymatic activity of hyperglycosylation of the yeast produced cellulases are discussed.

Influence of different glycosidic linkages on relative ion intensities in post‐source decay fragmentation of a xyloglucan heptaoligosaccharide using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Post-source decay fragment analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been applied to a highly branched xyloglucan heptasaccharide from tamarind seed. All fragment ions were produced by cleavage of the glycosidic linkages, including multi-site cleavages. The relative intensities of fragment ions that originated from one-site cleavages of the glycosidic linkages were much higher than those arising from two-site cleavages of the same kind of glycosidic linkage, which were in turn higher than those from three-site cleavages. The types of glycosidic linkages were an important factor which influenced the relative intensities of the MALDI-PSD (post-source decay) fragment ions. In the MALDI-PSD fragment spectrum of the xyloglucan heptasaccharide, the relative intensities of the ions produced by the cleavage of an alpha 1-6 glycosidic linkage were much higher than those arising from cleavage of the beta 1-4 glycosidic linkage.

Enzymatic synthesis and hydrolysis of xylogluco-oligosaccharides using the first archaeal α-xylosidase from Sulfolobus solfataricus

Abstract. The first, recently identified, archaeal α-xylosidase from Sulfolobus solfataricus (XylS) shows high specificity for hydrolysis of isoprimeverose [α-D-xylopyranosyl-(1,6)-D-glucopyranose, (X)], the p-nitrophenyl-β derivative of isoprimeverose, and xyloglucan oligosaccharides and has transxylosidic activity, forming, in a retaining mode, interesting α-xylosides. This article describes the synthesis of isoprimeverose, the disaccharidic repeating unit of xyloglucan, of the p-nitrophenyl-β derivative of isoprimeverose, and of a trisaccharide based on isoprimeverose that is one of the trisaccharidic building blocks of xyloglucan. A substrate structure–activity relationship is recognized for both the hydrolysis and the synthesis reactions of XylS, it being a biocatalyst (i) active hydrolytically only on X-ending substrates liberating a xylose molecule and (ii) capable of transferring xylose only on the nonreducing end glucose of p-nitrophenyl- (PNP)-β-D-cellobioside. The compounds synthesized by this enzyme are a starting point for enzymological studies of other new enzymes (i.e., xyloglucanases) for which suitable substrates are difficult to synthesize. This study also allows us to define the chemical characteristics of the xylose-transferring activity of this new archaeal enzyme, contributing to building up a library of different glycosidases with high specific selectivity for oligosaccharide synthesis.

Structural analysis of the oligosaccharide units of xyloglucan and their effects on growth of COLO 201 human tumor cells

Abstract The oligosaccharide units of xyloglucans from some fruits were analyzed by enzymatic digestion followed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The oligosaccharide units of the polysaccharides were XXXG, XXLG, XLXG, XXFG, XLLG, and XLFG [where each (1→4)-β-linked d -glucosyl residue in the backbone is given a one-letter code according to its substituents: G, β- d -Glc; X, α- d -Xyl-(1→6)-β- d -Glc; L, β- d -Gal-(1→2)-α- d -Xyl-(1→6)-β- d -Glc; F, α- l -Fuc-(1→2)-β- d -Gal-(1→2)-α- d -Xyl-(1→6)-β- d -Glc] in an approximate molar ratio of 10:51:4:13:11:11 for kiwi, of 23:3:40:27:2:5 for peach, of 31:1:9:19:1:39 for avocado, of 35:3:12:19:5:26 for apple, of 29:5:4:33:4:25 for fig, of 29:17:1:29:13:11 for pineapple, and of 27:4:8:21:4:36 for mandarin. In addition, different types of xyloglucan oligo- and polysaccharides were tested for effects on the growth of COLO 201 human tumor cells. The cell growth was reduced by fucose-containing oligo- and polysaccharides.

Crystallization and preliminary X‐ray crystallographic study on a xyloglucan‐specific exo‐β‐glycosidase, oligoxyloglucan reducing‐end specific cellobiohydrolase

A novel xyloglucan-specific exo-beta-glycosidase, oligoxyloglucan reducing-end specific cellobiohydrolase (OXG-RCBH), recognizes the reducing end of oligoxyloglucan and releases two glucosyl residue segments from the main chain. OXG-RCBH was crystallized by the hanging-drop vapour-diffusion method with polyethylene glycol 3000 and polyethylene glycol 400 as precipitants. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 61.0, b = 146.9, c = 211.9 A. The crystals diffract to a resolution of 2.2 A and are suitable for X-ray structure analysis.