Xylobiose treatment strengthens intestinal barrier function by regulating claudin 2 and heat shock protein 27 expression in human Caco-2 cells.

BACKGROUND: Xylobiose, a non-digestible disaccharide, largely contributes to the beneficial physiological effects of xylooligosaccharides. However, there is insufficient evidence to assess the direct effect of xylobiose on intestinal barrier function. Here, we investigated the intestinal barrier function in human intestinal Caco-2 cells treated with xylobiose. RESULTS: In total, 283 genes were upregulated and 256 genes were downregulated in xylobiose-treated Caco-2 cells relative to the controls. We focused on genes related to intestinal barrier function, such as tight junction (TJ) and heat shock protein (HSP). Xylobiose decreased the expression of the TJ gene Claudin 2 (CLDN2) and increased the expression of the cytoprotective HSP genes HSPB1 and HSPA1A, which encode HSP27 and HSP70, respectively. Immunoblot analysis confirmed that xylobiose suppressed CLDN2 expression and enhanced HSP27 and HSP70 expression. A quantitative reverse transcription-PCR and promoter assays indicated that xylobiose post-transcriptionally regulated CLDN2 and HSPB1 levels. Additionally, selective inhibition of phosphatidyl-3-inositol kinase (PI3K) inhibited xylobiose-mediated CLDN2 expression, whereas HSP27 expression induced by xylobiose was sensitive to the inhibition of PI3K, mitogen-activated protein kinase kinase and Src. CONCLUSION: The results of the present study reveal that xylobiose suppresses CLDN2 and increases HSP27 expression in intestinal Caco-2 cells via post-transcriptional regulation, potentially strengthening intestinal barrier integrity; however, these effects seem to occur via different signaling pathways. Our findings may help to assess the physiological role of xylobiose. © 2023 Society of Chemical Industry.

Crystal structure of reducing-end xylose-releasing exoxylanase in subfamily 7 of glycoside hydrolase family 30.

TcXyn30A from Talaromyces cellulolyticus, which belongs to subfamily 7 of the glycoside hydrolase family 30 (GH30-7), releases xylose from the reducing end of xylan and xylooligosaccharides (XOSs), the so-called reducing-end xylose-releasing exoxylanase (ReX). In this study, the crystal structures of TcXyn30A with and without xylose at subsite +1 (the binding site of the xylose residue at the reducing end) were determined. This is the first report on the structure of ReX in the family GH30-7. TcXyn30A forms a dimer. The complex structure of TcXyn30A with xylose revealed that subsite +1 is located at the dimer interface. TcXyn30A recognizes xylose at subsite +1 composed of amino acid residues from each monomer and blocks substrate binding to subsite +2 by dimer formation. Thus, the dimeric conformation is responsible for ReX activity. The structural comparison between TcXyn30A and the homologous enzyme indicated that subsite -2 is composed of assembled three stacked Trp residues, Trp49, Trp333, and Trp334, allowing TcXyn30A to accommodate xylan and any branched XOSs decorated with a substitution such as α-1,2-linked 4-O-methyl-d-glucuronic acid or α-1,2- and/or -1,3-linked L-arabinofuranose. These findings provide an insight into the structural determinants for ReX activity of TcXyn30A.

Crystal structure of metagenomic ß-glycosidase MeBglD2 in complex with various saccharides.

Metagenomic MeBglD2 is a glycoside hydrolase family 1 (GH1) ß-glycosidase that has ß-glucosidase, ß-fucosidase, and ß-galactosidase activities, and is highly activated in the presence of monosaccharides and disaccharides. The ß-glucosidase activity of MeBglD2 increases in a cellobiose concentration-dependent manner and is not inhibited by a high concentration of D-glucose or cellobiose. Previously, we solved the crystal structure of MeBglD2 and designed a thermostable mutant; however, the mechanism of substrate recognition of MeBglD2 remains poorly understood. In this paper, we report the X-ray crystal structures of MeBglD2 complexed with various saccharides, such as D-glucose, D-xylose, cellobiose, and maltose. The results showed that subsite - 1 of MeBglD2, which contained two catalytic glutamate residues (a nucleophilic Glu356 and an acid/base Glu170) was common to other GH1 enzymes, but the positive subsites (+ 1 and + 2) had different binding modes depending on the type of sugar. Three residues (Glu183, Asn227, and Asn229), located at the positive subsites of MeBglD2, were involved in substrate specificity toward cellobiose and/or chromogenic substrates in the presence of additive sugars. The docking simulation of MeBglD2-cellobiose indicated that Asn229 and Trp329 play important roles in the recognition of + 1 D-glucose in cellobiose. Our findings provide insights into the unique substrate recognition mechanism of GH1, which can incorporate a variety of saccharides into its positive subsites. KEY POINTS: • Metagenomic glycosidase, MeBglD2, recognizes various saccharides • Structures of metagenomic MeBglD2 complexed with various saccharides are determined • MeBglD2 has a unique substrate recognition mechanism at the positive subsites.

Characterization of an extracellular α-xylosidase involved in xyloglucan degradation in Aspergillus oryzae.

α-Xylosidases release the α-D-xylopyranosyl side chain from di- and oligosaccharides derived from xyloglucans and are involved in xyloglucan degradation. In this study, an extracellular α-xylosidase, named AxyB, is identified and characterized in Aspergillus oryzae. AxyB belongs to the glycoside hydrolase family 31 and releases D-xylose from isoprimeverose (α-D-xylopyranosyl-(1 → 6)-D-glucopyranose) and xyloglucan oligosaccharides. In the hydrolysis of xyloglucan oligosaccharides (XLLG, Glc4Xyl3Gal2 nonasaccharide; XLXG/XXLG, Glc4Xyl3Gal1 octasaccharide; and XXXG, Glc4Xyl3 heptasaccharide), AxyB releases one molecule of the xylopyranosyl side chain attached to the non-reducing end of the ß-1,4-glucan main chain of these xyloglucan oligosaccharides to yield GLLG (Glc4Xyl2Gal2), GLXG/GXLG (Glc4Xyl2Gal1), and GXXG (Glc4Xyl2). A. oryzae has both extracellular and intracellular α-xylosidase, suggesting that xyloglucan oligosaccharides are degraded by a combination of isoprimeverose-producing oligoxyloglucan hydrolase and intracellular α-xylosidase and a combination of extracellular α-xylosidase and ß-glucosidase(s) in A. oryzae. KEY POINTS: • An extracellular α-xylosidase, AxyB, is identified in Aspergillus oryzae. • AxyB releases the xylopyranosyl side chain from xyloglucan oligosaccharides. • Different sets of glycosidases degrade xyloglucan oligosaccharides in A. oryzae.

Crystal structure of glycoside hydrolase family 31 α-xylosidase from a soil metagenome.

MeXyl31, a member of glycoside hydrolase family 31 (GH31), is the α-xylosidase isolated from a soil metagenomic library. The enzyme degrades α-xylosyl substrate such as isoprimeverose, α-d-xylopyranosyl-(1→6)-glucopyranose. The crystal structure of MeXyl31 was determined at 1.80 Å resolution. MeXyl31 forms the tetrameric state. The complexed structure with a xylose in the -1 subsite (α-xylose binding site) shows that the enzyme strictly recognizes α-xylose. Structural comparison between MeXyl31 and its homologue, Aspergillus niger α-xylosidase in GH31, gave insights into the positive subsite of MeXyl31. First, in the tetrameric enzyme, two monomers (a catalytic monomer and the adjacent monomer), are involved in substrate recognition. Second, the adjacent monomer composes a part of positive subsites in MeXyl31. Docking simulation and site-directed mutagenesis suggested that the Arg100 from the adjacent monomer is partially involved in the recognizing of a glucopyranose of isoprimeverose.

Structural and functional characterization of a bifunctional GH30-7 xylanase B from the filamentous fungus Talaromyces cellulolyticus.

Glucuronoxylanases are endo-xylanases and members of the glycoside hydrolase family 30 subfamilies 7 (GH30-7) and 8 (GH30-8). Unlike for the well-studied GH30-8 enzymes, the structural and functional characteristics of GH30-7 enzymes remain poorly understood. Here, we report the catalytic properties and three-dimensional structure of GH30-7 xylanase B (Xyn30B) identified from the cellulolytic fungus Talaromyces cellulolyticus Xyn30B efficiently degraded glucuronoxylan to acidic xylooligosaccharides (XOSs), including an α-1,2-linked 4-O-methyl-d-glucuronosyl substituent (MeGlcA). Rapid analysis with negative-mode electrospray-ionization multistage MS (ESI(-)-MS n ) revealed that the structures of the acidic XOS products are the same as those of the hydrolysates (MeGlcA2Xyl n , n > 2) obtained with typical glucuronoxylanases. Acidic XOS products were further degraded by Xyn30B, releasing first xylobiose and then xylotetraose and xylohexaose as transglycosylation products. This hydrolase reaction was unique to Xyn30B, and the substrate was cleaved at the xylobiose unit from its nonreducing end, indicating that Xyn30B is a bifunctional enzyme possessing both endo-glucuronoxylanase and exo-xylobiohydrolase activities. The crystal structure of Xyn30B was determined as the first structure of a GH30-7 xylanase at 2.25 Å resolution, revealing that Xyn30B is composed of a pseudo-(α/ß)8-catalytic domain, lacking an α6 helix, and a small ß-rich domain. This structure and site-directed mutagenesis clarified that Arg46, conserved in GH30-7 glucuronoxylanases, is a critical residue for MeGlcA appendage-dependent xylan degradation. The structural comparison between Xyn30B and the GH30-8 enzymes suggests that Asn93 in the ß2-α2 loop is involved in xylobiohydrolase activity. In summary, our findings indicate that Xyn30B is a bifunctional endo- and exo-xylanase.

Identification and characterization of two xyloglucan-specific endo-1,4-glucanases in Aspergillus oryzae.

Aspergillus oryzae produces glycoside hydrolases to degrade xyloglucan. We identified and characterized two xyloglucan-specific endo-1,4-glucanases (xyloglucanases) named Xeg12A and Xeg5A. Based on their amino acid sequences, Xeg12A and Xeg5A were classified into glycoside hydrolase families GH12 and GH5, respectively. Xeg12A degrades tamarind seed xyloglucan polysaccharide into xyloglucan oligosaccharides containing four glucopyranosyl residues as main chains, including heptasaccharides (XXXG: Glc4Xyl3), octasaccharides (XXLG and XLXG: Glc4Xyl3Gal1), and nonasaccharides (XLLG: Glc4Xyl3Gal2). By contrast, Xeg5A produces various xyloglucan oligosaccharides from xyloglucan. Xeg5A hydrolyzes xyloglucan into not only XXXG, XXLG/XLXG, and XLLG but also disaccharides (isoprimeverose: Glc1Xyl1), tetrasaccharides (XX: Glc2Xyl2 and LG: Glc2Xyl1Gal1), and so on. Xeg12A is a typical endo-dissociative-type xyloglucanase that repeats hydrolysis and desorption from xyloglucan. Conversely, Xeg5A acts as an endo-processive-type xyloglucanase that hydrolyzes xyloglucan progressively without desorption. These results indicate that although both Xeg12A and Xeg5A contribute to the degradation of xyloglucan, they have different modes of activity toward xyloglucan, and the hydrolysis machinery of Xeg5A is unique compared with that of other known GH5 enzymes. KEY POINTS: • We identified two xyloglucanases, Xeg12A and Xeg5A, in A. oryzae. • Modes of activity and regiospecificities of Xeg12A and Xeg5A were clearly different. • Xeg5A is a unique xyloglucanase that produces low-molecular-weight oligosaccharides.

Crystal structure and substrate recognition mechanism of Aspergillus oryzae isoprimeverose-producing enzyme.

Isoprimeverose-producing enzymes (IPases) release isoprimeverose (α-d-xylopyranosyl-(1 → 6)-d-glucopyranose) from the non-reducing end of xyloglucan oligosaccharides. Aspergillus oryzae IPase (IpeA) is classified as a member of the glycoside hydrolase family 3 (GH3); however, it has unusual substrate specificity compared with other GH3 enzymes. Xylopyranosyl branching at the non-reducing ends of xyloglucan oligosaccharides is vital for IpeA activity. We solved the crystal structure of IpeA with isoprimeverose at 2.4 Šresolution, showing that the structure of IpeA formed a dimer and was composed of three domains: an N-terminal (ß/α)8 TIM-barrel domain, α/ß/α sandwich fold domain, and a C-terminal fibronectin-like domain. The catalytic TIM-barrel domain possessed a catalytic nucleophile (Asp300) and acid/base (Glu524) residues. Interestingly, we found that the cavity of the active site of IpeA was larger than that of other GH3 enzymes, and subsite -1' played an important role in its activity. The glucopyranosyl and xylopyranosyl residues of isoprimeverose were located at subsites -1 and -1', respectively. Gln58 and Tyr89 contributed to the interaction with the xylopyranosyl residue of isoprimeverose through hydrogen bonding and stacking effects, respectively. Our findings provide new insights into the substrate recognition of GH3 enzymes.

GH30-7 Endoxylanase C from the Filamentous Fungus Talaromyces cellulolyticus.

Glycoside hydrolase family 30 subfamily 7 (GH30-7) enzymes include various types of xylanases, such as glucuronoxylanase, endoxylanase, xylobiohydrolase, and reducing-end xylose-releasing exoxylanase. Here, we characterized the mode of action and gene expression of the GH30-7 endoxylanase from the cellulolytic fungus Talaromyces cellulolyticus (TcXyn30C). TcXyn30C has a modular structure consisting of a GH30-7 catalytic domain and a C-terminal cellulose binding module 1, whose cellulose-binding ability has been confirmed. Sequence alignment of GH30-7 xylanases exhibited that TcXyn30C has a conserved Phe residue at the position corresponding to a conserved Arg residue in GH30-7 glucuronoxylanases, which is required for the recognition of the 4-O-methyl-α-d-glucuronic acid (MeGlcA) substituent. TcXyn30C degraded both glucuronoxylan and arabinoxylan with similar kinetic constants and mainly produced linear xylooligosaccharides (XOSs) with 2 to 3 degrees of polymerization, in an endo manner. Notably, the hydrolysis of glucuronoxylan caused an accumulation of 22-(MeGlcA)-xylobiose (U4m2X). The production of this acidic XOS is likely to proceed via multistep reactions by putative glucuronoxylanase activity that produces 22-(MeGlcA)-XOSs (X n U4m2X, n ≥ 0) in the initial stages of the hydrolysis and by specific release of U4m2X from a mixture containing X n U4m2X. Our results suggest that the unique endoxylanase activity of TcXyn30C may be applicable to the production of linear and acidic XOSs. The gene xyn30C was located adjacent to the putative GH62 arabinofuranosidase gene (abf62C) in the T. cellulolyticus genome. The expression of both genes was induced by cellulose. The results suggest that TcXyn30C may be involved in xylan removal in the hydrolysis of lignocellulose by the T. cellulolyticus cellulolytic system.IMPORTANCE Xylooligosaccharides (XOSs), which are composed of xylose units with a ß-1,4 linkage, have recently gained interest as prebiotics in the food and feed industry. Apart from linear XOSs, branched XOSs decorated with a substituent such as methyl glucuronic acid and arabinose also have potential applications. Endoxylanase is a promising tool in producing XOSs from xylan. The structural variety of XOSs generated depends on the substrate specificity of the enzyme as well as the distribution of the substituents in xylan. Thus, the exploration of endoxylanases with novel specificities is expected to be useful in the provision of a series of XOSs. In this study, the endoxylanase TcXyn30C from Talaromyces cellulolyticus was characterized as a unique glycoside hydrolase belonging to the family GH30-7, which specifically releases 22-(4-O-methyl-α-d-glucuronosyl)-xylobiose from hardwood xylan. This study provides new insights into the production of linear and branched XOSs by GH30-7 endoxylanase.

Mode of Action of GH30-7 Reducing-End Xylose-Releasing Exoxylanase A (Xyn30A) from the Filamentous Fungus Talaromyces cellulolyticus.

In this study, we characterized the mode of action of reducing-end xylose-releasing exoxylanase (Rex), which belongs to the glycoside hydrolase family 30-7 (GH30-7). GH30-7 Rex, isolated from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), exists as a dimer. The purified Xyn30A released xylose from linear xylooligosaccharides (XOSs) 3 to 6 xylose units in length with similar kinetic constants. Hydrolysis of branched, borohydride-reduced, and p-nitrophenyl XOSs clarified that Xyn30A possesses a Rex activity. 1H nuclear magnetic resonance (1H NMR) analysis of xylotriose hydrolysate indicated that Xyn30A degraded XOSs via a retaining mechanism and without recognizing an anomeric structure at the reducing end. Hydrolysis of xylan by Xyn30A revealed that the enzyme continuously liberated both xylose and two types of acidic XOSs: 22-(4-O-methyl-α-d-glucuronyl)-xylotriose (MeGlcA2Xyl3) and 22-(MeGlcA)-xylobiose (MeGlcA2Xyl2). These acidic products were also detected during hydrolysis using a mixture of MeGlcA2Xyl n (n = 2 to 14) as the substrate. This indicates that Xyn30A can release MeGlcA2Xyl n (n = 2 and 3) in an exo manner. Comparison of subsites in Xyn30A and GH30-7 glucuronoxylanase using homology modeling suggested that the binding of the reducing-end residue at subsite +2 was partially prevented by a Gln residue conserved in GH30-7 Rex; additionally, the Arg residue at subsite -2b, which is conserved in glucuronoxylanase, was not found in Xyn30A. Our results lead us to propose that GH30-7 Rex plays a complementary role in hydrolysis of xylan by fungal cellulolytic systems.IMPORTANCE Endo- and exo-type xylanases depolymerize xylan and play crucial roles in the assimilation of xylan in bacteria and fungi. Exoxylanases release xylose from the reducing or nonreducing ends of xylooligosaccharides; this is generated by the activity of endoxylanases. ß-Xylosidase, which hydrolyzes xylose residues on the nonreducing end of a substrate, is well studied. However, the function of reducing-end xylose-releasing exoxylanases (Rex), especially in fungal cellulolytic systems, remains unclear. This study revealed the mode of xylan hydrolysis by Rex from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), which belongs to the glycoside hydrolase family 30-7 (GH30-7). A conserved residue related to Rex activity is found in the substrate-binding site of Xyn30A. These findings will enhance our understanding of the function of GH30-7 Rex in the cooperative hydrolysis of xylan by fungal enzymes.

Crystal structure of a bacterial unsaturated glucuronyl hydrolase with specificity for heparin.

Extracellular matrix molecules such as glycosaminoglycans (GAGs) are typical targets for some pathogenic bacteria, which allow adherence to host cells. Bacterial polysaccharide lyases depolymerize GAGs in ß-elimination reactions, and the resulting unsaturated disaccharides are subsequently degraded to constituent monosaccharides by unsaturated glucuronyl hydrolases (UGLs). UGL substrates are classified as 1,3- and 1,4-types based on the glycoside bonds. Unsaturated chondroitin and heparin disaccharides are typical members of 1,3- and 1,4-types, respectively. Here we show the reaction modes of bacterial UGLs with unsaturated heparin disaccharides by x-ray crystallography, docking simulation, and site-directed mutagenesis. Although streptococcal and Bacillus UGLs were active on unsaturated heparin disaccharides, those preferred 1,3- rather than 1,4-type substrates. The genome of GAG-degrading Pedobacter heparinus encodes 13 UGLs. Of these, Phep_2830 is known to be specific for unsaturated heparin disaccharides. The crystal structure of Phep_2830 was determined at 1.35-Å resolution. In comparison with structures of streptococcal and Bacillus UGLs, a pocket-like structure and lid loop at subsite +1 are characteristic of Phep_2830. Docking simulations of Phep_2830 with unsaturated heparin disaccharides demonstrated that the direction of substrate pyranose rings differs from that in unsaturated chondroitin disaccharides. Acetyl groups of unsaturated heparin disaccharides are well accommodated in the pocket at subsite +1, and aromatic residues of the lid loop are required for stacking interactions with substrates. Thus, site-directed mutations of the pocket and lid loop led to significantly reduced enzyme activity, suggesting that the pocket-like structure and lid loop are involved in the recognition of 1,4-type substrates by UGLs.

Crystal structure of Pedobacter heparinus heparin lyase Hep III with the active site in a deep cleft.

Pedobacter heparinus (formerly known as Flavobacterium heparinum) is a typical glycosaminoglycan-degrading bacterium that produces three heparin lyases, Hep I, Hep II, and Hep III, which act on heparins with 1,4-glycoside bonds between uronate and amino sugar residues. Being different from Hep I and Hep II, Hep III is specific for heparan sulfate. Here we describe the crystal structure of Hep III with the active site located in a deep cleft. The X-ray crystallographic structure of Hep III was determined at 2.20 Å resolution using single-wavelength anomalous diffraction. This enzyme comprised an N-terminal α/α-barrel domain and a C-terminal antiparallel ß-sheet domain as its basic scaffold. Overall structures of Hep II and Hep III were similar, although Hep III exhibited an open form compared with the closed form of Hep II. Superimposition of Hep III and heparin tetrasaccharide-bound Hep II suggested that an active site of Hep III was located in the deep cleft at the interface between its two domains. Three mutants (N240A, Y294F, and H424A) with mutations at the active site had significantly reduced enzyme activity. This is the first report of the structure-function relationship of P. heparinus Hep III.

Isopropanol production via the thermophilic bioconversion of sugars and syngas using metabolically engineered Moorella thermoacetica.

BACKGROUND: Isopropanol (IPA) is a commodity chemical used as a solvent or raw material for polymeric products, such as plastics. Currently, IPA production depends largely on high-CO2-emission petrochemical methods that are not sustainable. Therefore, alternative low-CO2 emission methods are required. IPA bioproduction using biomass or waste gas is a promising method. RESULTS: Moorella thermoacetica, a thermophilic acetogenic microorganism, was genetically engineered to produce IPA. A metabolic pathway related to acetone reduction was selected, and acetone conversion to IPA was achieved via the heterologous expression of secondary alcohol dehydrogenase (sadh) in the thermophilic bacterium. sadh-expressing strains were combined with acetone-producing strains, to obtain an IPA-producing strain. The strain produced IPA as a major product using hexose and pentose sugars as substrates (81% mol-IPA/mol-sugar). Furthermore, IPA was produced from CO, whereas acetate was an abundant byproduct. Fermentation using syngas containing both CO and H2 resulted in higher IPA production at the specific rate of 0.03 h-1. The supply of reducing power for acetone conversion from the gaseous substrates was examined by supplementing acetone to the culture, and the continuous and rapid conversion of acetone to IPA showed a sufficient supply of NADPH for Sadh. CONCLUSIONS: The successful engineering of M. thermoacetica resulted in high IPA production from sugars. M. thermoacetica metabolism showed a high capacity for acetone conversion to IPA in the gaseous substrates, indicating acetone production as the bottleneck in IPA production for further improving the strain. This study provides a platform for IPA production via the metabolic engineering of thermophilic acetogens.

Genetic engineering of a thermophilic acetogen, Moorella thermoacetica Y72, to enable acetoin production.

Acetogens are among the key microorganisms involved in the bioproduction of commodity chemicals from diverse carbon resources, such as biomass and waste gas. Thermophilic acetogens are particularly attractive because fermentation at higher temperatures offers multiple advantages. However, the main target product is acetic acid. Therefore, it is necessary to reshape metabolism using genetic engineering to produce the desired chemicals with varied carbon lengths. Although such metabolic engineering has been hampered by the difficulty involved in genetic modification, a model thermophilic acetogen, M. thermoacetica ATCC 39073, is the case with a few successful cases of C2 and C3 compound production, other than acetate. This brief report attempts to expand the product spectrum to include C4 compounds by using strain Y72 of Moorella thermoacetica. Strain Y72 is a strain related to the type strain ATCC 39073 and has been reported to have a less stringent restriction-modification system, which could alleviate the cumbersome transformation process. A simplified procedure successfully introduced a key enzyme for acetoin (a C4 chemical) production, and the resulting strains produced acetoin from sugars and gaseous substrates. The culture profile revealed varied acetoin yields depending on the type of substrate and culture conditions, implying the need for further engineering in the future. Thus, the use of a user-friendly chassis could benefit the genetic engineering of M. thermoacetica.

Identification and Characterization of Novel Intracellular α-Xylosidase in Aspergillus oryzae.

α-Xylosidase releases xylopyranosyl side chains from xyloglucan oligosaccharides and is vital for xyloglucan degradation. Previously, we identified and characterized two α-xylosidases, intracellular AxyA and extracellular AxyB, in Aspergillus oryzae. In this study, we identified a third α-xylosidase, termed AxyC, in A. oryzae. These three A. oryzae α-xylosidases belong to the glycoside hydrolase family 31, but there are clear differences in substrate specificity. Both AxyA and AxyB showed much higher hydrolytic activity toward isoprimeverose (α-D-xylopyranosyl-1,6-glucose) than p-nitrophenyl α-D-xylopyranoside. In contrast, the specific activity of AxyC toward the p-nitrophenyl substrate was approximately 950-fold higher than that toward isoprimeverose. Our study revealed that there are multiple α-xylosidases with different substrate specificities in A. oryzae.

Structural identification of catalytic His158 of PtMAC2p from Pseudozyma tsukubaensis, an acyltransferase involved in mannosylerythritol lipids formation.

Mannosylerythritol lipids (MELs) are extracellular glycolipids produced by the basidiomycetous yeast strains. MELs consist of the disaccharide mannosylerythritol, which is acylated with fatty acids and acetylated at the mannose moiety. In the MEL biosynthesis pathway, an acyltransferase from Pseudozyma tsukubaensis, PtMAC2p, a known excellent MEL producer, has been identified to catalyze the acyl-transfer of fatty acid to the C3'-hydroxyl group of mono-acylated MEL; however, its structure remains unclear. Here, we performed X-ray crystallography of recombinant PtMAC2p produced in Escherichia coli and homogeneously purified it with catalytic activity in vitro. The crystal structure of PtMAC2p was determined by single-wavelength anomalous dispersion using iodide ions. The crystal structure shows that PtMAC2p possesses a large putative catalytic tunnel at the center of the molecule. The structural comparison demonstrated that PtMAC2p is homologous to BAHD acyltransferases, although its amino acid-sequence identity was low (<15%). Interestingly, the HXXXD motif, which is a conserved catalytic motif in the BAHD acyltransferase superfamily, is partially conserved as His158-Thr159-Leu160-Asn161-Gly162 in PtMAC2p, i.e., D in the HXXXD motif is replaced by G in PtMAC2p. Site-directed mutagenesis of His158 to Ala resulted in more than 1,000-fold decrease in the catalytic activity of PtMAC2p. These findings suggested that His158 in PtMAC2p is the catalytic residue. Moreover, in the putative catalytic tunnel, hydrophobic amino acid residues are concentrated near His158, suggesting that this region is a binding site for the fatty acid side chain of MEL (acyl acceptor) and/or acyl-coenzyme A (acyl donor). To our knowledge, this is the first study to provide structural insight into the catalytic activity of an enzyme involved in MEL biosynthesis.

Mechanistic insights into Schizosaccharomyces pombe GT-A family protein Pvg3 in the biosynthesis of pyruvylated ß1,3-galactose of N-linked oligosaccharides.

N-linked oligosaccharides in the fission yeast Schizosaccharomyces pombe contain large amounts of d-galactose (Gal), which mainly comprises α1,2- and α1,3-linked Gal except for pyruvylated ß1,3-linked Gal (PvGalß) at the non-reducing end. The PvGalß unit of N-glycans is important for regulating nonsexual flocculation and invasive growth, but the mechanistic basis for ß-galactosylation in fission yeast is poorly understood. To gain insight into this mechanism, we have characterized three genes previously identified to be involved in PvGalß biosynthesis (pvg2, pvg3, and pvg5), with a focus on pvg3, which is predicted to contain a domain conserved in galactosyltransferase family 31 (GT31) proteins. Fluorescent microscopy revealed that Pvg3 is stably localized at the Golgi membrane, regardless of the presence of pvg2+ or pvg5+, suggesting that Pvg2 and Pvg5 are essential for the function of Pvg3 as a ß1,3-galactosyltransferase, and not for its localization to the Golgi. Mutation of the GT31 family DXD motif and GT-A fold in Pvg3 resulted in loss of catalytic activity in vivo, supporting the idea that Pvg3 is a GT-A type ß1,3-galactosyltransferase. Docking simulations further indicated that Pvg3 can recognize donor and acceptor substrates suitable for ß-(1→3) bond formation. Yeast two-hybrid assay showed that Pvg5 physically interacts with Pvg3 and the pyruvyltransferase Pvg1. Collectively, these results provide insight into ß-galactosylation catalyzed by Pvg3 and the supporting role of Pvg5 in PvGalß biosynthesis.

Structural basis for the allosteric pathway of 4-amino-4-deoxychorismate synthase.

4-Amino-4-deoxychorismate synthase (ADCS), a chorismate-utilizing enzyme, is composed of two subunits: PabA and PabB. PabA is a glutamine amidotransferase that hydrolyzes glutamine into glutamate and ammonia. PabB is an aminodeoxychorismate synthase that converts chorismate to 4-amino-4-deoxychorismate (ADC) using the ammonia produced by PabA. ADCS functions under allosteric regulation between PabA and PabB. However, the allosteric mechanism remains unresolved because the structure of the PabA-PabB complex has not been determined. Here, the crystal structure and characterization of PapA from Streptomyces venezuelae (SvPapA), a bifunctional enzyme comprising the PabA and PabB domains, is reported. SvPapA forms a unique dimer in which PabA and PabB domains from different monomers complement each other and form an active structure. The chorismate-bound structure revealed that recognition of the C1 carboxyl group by Thr501 and Gly502 of the 498-PIKTG-502 motif in the PabB domain is essential for the catalytic Lys500 to reach the C2 atom, a reaction-initiation site. SvPapA demonstrated ADCS activity in the presence of Mg2+ when glutamate or NH+4 was used as the amino donor. The crystal structure indicated that the Mg2+-binding position changed depending on the binding of chorismate. In addition, significant structural changes were observed in the PabA domain depending on the presence or absence of chorismate. This study provides insights into the structural factors that are involved in the allosteric regulation of ADCS.

Structural determinants in streptococcal unsaturated glucuronyl hydrolase for recognition of glycosaminoglycan sulfate groups.

Pathogenic Streptococcus agalactiae produces polysaccharide lyases and unsaturated glucuronyl hydrolase (UGL), which are prerequisite for complete degradation of mammalian extracellular matrices, including glycosaminoglycans such as chondroitin and hyaluronan. Unlike the Bacillus enzyme, streptococcal UGLs prefer sulfated glycosaminoglycans. Here, we show the loop flexibility for substrate binding and structural determinants for recognition of glycosaminoglycan sulfate groups in S. agalactiae UGL (SagUGL). UGL also degraded unsaturated heparin disaccharides; this indicates that the enzyme released unsaturated iduronic and glucuronic acids from substrates. We determined the crystal structures of SagUGL wild-type enzyme and both substrate-free and substrate-bound D175N mutants by x-ray crystallography and noted that the loop over the active cleft exhibits flexible motion for substrate binding. Several residues in the active cleft bind to the substrate, unsaturated chondroitin disaccharide with a sulfate group at the C-6 position of GalNAc residue. The sulfate group is hydrogen-bonded to Ser-365 and Ser-368 and close to Lys-370. As compared with wild-type enzyme, S365H, S368G, and K370I mutants exhibited higher Michaelis constants toward the substrate. The conversion of SagUGL to Bacillus sp. GL1 UGL-like enzyme via site-directed mutagenesis demonstrated that Ser-365 and Lys-370 are essential for direct binding and for electrostatic interaction, respectively, for recognition of the sulfate group by SagUGL. Molecular conversion was also achieved in SagUGL Arg-236 with an affinity for the sulfate group at the C-4 position of the GalNAc residue. These residues binding to sulfate groups are frequently conserved in pathogenic bacterial UGLs, suggesting that the motif "R-//-SXX(S)XK" (where the hyphen and slash marks in the motif indicate the presence of over 100 residues in the enzyme and parentheses indicate that Ser-368 makes little contribution to enzyme activity) is crucial for degradation of sulfated glycosaminoglycans.

Structural basis for the catalytic mechanism of the glycoside hydrolase family 3 isoprimeverose-producing oligoxyloglucan hydrolase from Aspergillus oryzae.

Aspergillus oryzae isoprimeverose-producing oligoxyloglucan hydrolase (IpeA) releases isoprimeverose units (α-d-xylopyranosyl-(1→6)-d-glucose) from the non-reducing end of xyloglucan oligosaccharides and belongs to glycoside hydrolase family 3. In this paper, we report the X-ray crystal structure of the IpeA complexed with a xyloglucan oligosaccharide, (XXXG: Glc4 Xyl3 ). Trp515 of IpeA plays a critical role in XXXG recognition at positive subsites. In addition, docking simulation of IpeA-XXXG suggested that two Tyr residues (Tyr268 and Tyr445) are involved in the catalytic reaction mechanism of IpeA. Tyr268 plays an important role in product turnover, whereas Tyr445 stabilizes the acid/base Glu524 residue, which serves as a proton donor. Our findings indicate that the substrate recognition machinery of IpeA is specifically adapted to xyloglucan oligosaccharides.