Katsuro Yaoi

Aminopeptidase N isoforms from the midgut of Bombyx mori and Plutella xylostella -- their classification and the factors that determine their binding specificity to Bacillus thuringiensis Cry1A toxin.

Novel aminopeptidase N (APN) isoform cDNAs, BmAPN3 and PxAPN3, from the midguts of Bombyx mori and Plutella xylostella, respectively, were cloned, and a total of eight APN isoforms cloned from B. mori and P. xylostella were classified into four classes. Bacillus thuringiensis Cry1Aa and Cry1Ab toxins were found to bind to specific APN isoforms from the midguts of B. mori and P. xylostella, and binding occurred with fragments that corresponded to the BmAPN1 Cry1Aa toxin‐binding region of each APN isoform. The results suggest that APN isoforms have a common toxin‐binding region, and that the apparent specificity of Cry1Aa toxin binding to each intact APN isoform seen in SDS–PAGE is determined by factors such as expression level in conjunction with differences in binding affinity.

Characterization of a Novel β-Glucosidase from a Compost Microbial Metagenome with Strong Transglycosylation Activity

Background: There is an ongoing search for a β-glucosidase that has insensitivity to product inhibition. Results: A β-glucosidase, Td2F2, was isolated from a metagenomic library and has high glucose tolerance, and its activity is enhanced by various monosaccharides. Conclusion: The high glucose tolerance of Td2F2 is related to high transglycosylation activity. Significance: This is the first report of a β-glucosidase having high glucose tolerance and high transglycosylation activity. The β-glucosidase encoded by the td2f2 gene was isolated from a compost microbial metagenomic library by functional screening. The protein was identified to be a member of the glycoside hydrolase family 1 and was overexpressed in Escherichia coli, purified, and biochemically characterized. The recombinant β-glucosidase, Td2F2, exhibited enzymatic activity with β-glycosidic substrates, with preferences for glucose, fucose, and galactose. Hydrolysis occurred at the nonreducing end and in an exo manner. The order of catalytic efficiency for glucodisaccharides and cellooligosaccharides was sophorose > cellotetraose > cellotriose > laminaribiose > cellobiose > cellopentaose > gentiobiose, respectively. Intriguingly, the p-nitrophenyl-β-d-glucopyranoside hydrolysis activity of Td2F2 was activated by various monosaccharides and sugar alcohols. At a d-glucose concentration of 1000 mm, enzyme activity was 6.7-fold higher than that observed in the absence of d-glucose. With 31.3 mm d-glucose, Td2F2 catalyzed transglycosylation to generate sophorose, laminaribiose, cellobiose, and gentiobiose. Transglycosylation products were detected under all activated conditions, suggesting that the activity enhancement induced by monosaccharides and sugar alcohols may be due to the transglycosylation activity of the enzyme. These results show that Td2F2 obtained from a compost microbial metagenome may be a potent candidate for industrial applications.

Generation and structural validation of a library of diverse xyloglucan-derived oligosaccharides, including an update on xyloglucan nomenclature

Xyloglucans are structurally complex plant cell wall polysaccharides that are involved in cell growth and expansion, energy metabolism, and signaling. Determining the structure-function relationships of xyloglucans would benefit from the availability of a comprehensive and structurally diverse collection of rigorously characterized xyloglucan oligosaccharides. Here, we present a workflow for the semi-preparative scale generation and purification of neutral and acidic xyloglucan oligosaccharides using a combination of enzymatic and chemical treatments and size-exclusion chromatography. Twenty-six of these oligosaccharides were purified to near homogeneity and their structures validated using a combination of matrix-assisted laser desorption/ionization mass spectrometry, high-performance anion exchange chromatography, and 1H nuclear magnetic resonance spectroscopy. Mass spectrometry and analytical chromatography were compared as methods for xyloglucan oligosaccharide quantification. 1H chemical shifts were assigned using two-dimensional correlation spectroscopy. A comprehensive update of the nomenclature describing xyloglucan side-chain structures is provided for reference.

A cadherin-like protein functions as a receptor for Bacillus thuringiensis Cry1Aa and Cry1Ac toxins on midgut epithelial cells of Bombyx mori larvae

Aminopeptidase N (APN) and cadherin‐like protein (BtR175) from Bombyx mori larvae were examined for their roles in Cry1Aa‐ and Cry1Ac‐induced lysis of B. mori midgut epithelial cells (MECs). APNs and BtR175 were present in all areas of the midgut, were particularly abundant in the posterior region, and were found only on columnar cell microvilli and not on the lateral membrane that makes cell–cell contacts. This distribution was in accordance with the distribution of Cry1A‐susceptible MECs in the midgut. The lytic activity of Cry1Aa and Cry1Ac on collagenase‐dissociated MECs was linearly dependent on toxin concentration. Although pre‐treatment of MECs with anti‐BtR175 antibody was observed to partially inhibit the lytic activity exerted by 0.1–1 nM Cry1Aa toxin or 5 nM Cry1Ac toxin, no significant inhibition was observed when MECs were pre‐treated with anti‐APN antibody. These results suggest that BtR175 functions as a major receptor for Cry1A toxins in the midgut of B. mori larvae.

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.

Engineering the Oryza sativa cell wall with rice NAC transcription factors regulating secondary wall formation

Plant tissues that require structural rigidity synthesize a thick, strong secondary cell wall of lignin, cellulose and hemicelluloses in a complicated bridged structure. Master regulators of secondary wall synthesis were identified in dicots, and orthologs of these regulators have been identified in monocots, but regulation of secondary cell wall formation in monocots has not been extensively studied. Here we demonstrate that the rice transcription factors SECONDARY WALL NAC DOMAIN PROTEINs (SWNs) can regulate secondary wall formation in rice (Oryza sativa) and are potentially useful for engineering the monocot cell wall. The OsSWN1 promoter is highly active in sclerenchymatous cells of the leaf blade and less active in xylem cells. By contrast, the OsSWN2 promoter is highly active in xylem cells and less active in sclerenchymatous cells. OsSWN2 splicing variants encode two proteins; the shorter protein (OsSWN2S) has very low transcriptional activation ability, but the longer protein (OsSWN2L) and OsSWN1 have strong transcriptional activation ability. In rice, expression of an OsSWN2S chimeric repressor, driven by the OsSWN2 promoter, resulted in stunted growth and para-wilting (leaf rolling and browning under normal water conditions) due to impaired vascular vessels. The same OsSWN2S chimeric repressor, driven by the OsSWN1 promoter, caused a reduction of cell wall thickening in sclerenchymatous cells, a drooping leaf phenotype, reduced lignin and xylose contents and increased digestibility as forage. These data suggest that OsSWNs regulate secondary wall formation in rice and manipulation of OsSWNs may enable improvements in monocotyledonous crops for forage or biofuel applications.

Substrate recognition by glycoside hydrolase family 74 xyloglucanase from the basidiomycete Phanerochaete chrysosporium.

The basidiomycete Phanerochaete chrysosporium produces xyloglucanase Xgh74B, which has the glycoside hydrolase (GH) family 74 catalytic domain and family 1 carbohydrate‐binding module, in cellulose‐grown culture. The recombinant enzyme, which was heterologously expressed in the yeast Pichia pastoris, had high hydrolytic activity toward xyloglucan from tamarind seed (TXG), whereas other β‐1,4‐glucans examined were poor substrates for the enzyme. The existence of the carbohydrate‐binding module significantly affects adsorption of the enzyme on crystalline cellulose, but has no effect on the hydrolysis of xyloglucan, indicating that the domain may contribute to the localization of the enzyme. HPLC and MALDI‐TOF MS analyses of the hydrolytic products of TXG clearly indicated that Xgh74B hydrolyzes the glycosidic bonds of unbranched glucose residues, like other GH family 74 xyloglucanases. However, viscometric analysis suggested that Xgh74B hydrolyzes TXG in a different manner from other known GH family 74 xyloglucanases. Gel permeation chromatography showed that Xgh74B initially produced oligosaccharides of degree of polymerization (DP) 16–18, and these oligosaccharides were then slowly hydrolyzed to final products of DP 7–9. In addition, the ratio of oligosaccharides of DP 7–9 versus those of DP 16–18 was dependent upon the pH of the reaction mixture, indicating that the affinity of Xgh74B for the oligosaccharides of DP 16–18 is affected by the ionic environment at the active site.

The crystal structure of a xyloglucan‐specific endo‐β‐1,4‐glucanase from Geotrichum sp. M128 xyloglucanase reveals a key amino acid residue for substrate specificity

Geotrichum sp. M128 possesses two xyloglucan‐specific glycoside hydrolases belonging to family 74, xyloglucan‐specific endo‐β‐1,4‐glucanase (XEG) and oligoxyloglucan reducing‐end‐specific cellobiohydrolase (OXG‐RCBH). Despite their similar amino acid sequences (48% identity), their modes of action and substrate specificities are distinct. XEG catalyzes the hydrolysis of xyloglucan polysaccharides in endo mode, while OXG‐RCBH acts on xyloglucan oligosaccharides at the reducing end in exo mode. Here, we determined the crystal structure of XEG at 2.5 Å resolution, and compared it to a previously determined structure of OXG‐RCBH. For the most part, the amino acid residues that interact with substrate are conserved between the two enzymes. However, there are notable differences at subsite positions −1 and +2. OXG‐RCBH has a loop around the +2 site that blocks one end of the active site cleft, which accounts for its exo mode of action. In contrast, XEG lacks a corresponding loop at this site, thereby allowing binding to the middle of the main chain of the substrate. At the −1 site in OXG‐RCBH, Asn488 interacts with the xylose side chain of the substrate, whereas the −1 site is occupied by Tyr457 in XEG. To confirm the contribution of this residue to substrate specificity, Tyr457 was substituted by Gly in XEG. The wild‐type XEG cleaved the oligoxyloglucan at a specific site; the Y457G variant cleaved the same substrate, but at various sites. Together, the absence of a loop in the cleft and the presence of bulky Tyr457 determine the substrate specificity of XEG.