Structural and mutational analysis of glycoside hydrolase family 1 Br2 ß-glucosidase derived from bovine rumen metagenome.

Ruminant animals rely on the activities of ß-glucosidases from residential microbes to convert feed fibers into glucose for further metabolic uses. In this report, we determined the structures of Br2, which is a glycoside hydrolase family 1 ß-glucosidase from the bovine rumen metagenome. Br2 folds into a classical (ß/α)8-TIM barrel domain but displays unique structural features at loop ß5→α5 and α-helix 5, resulting in different positive subsites from those of other GH1 enzymes. Br2 exhibited the highest specificity toward laminaritriose, suggesting its involvement in ß-glucan hydrolysis in digested feed. We then substituted the residues at subsites +1 and + 2 of Br2 with those of Halothermothrix orenii ß-glucosidase. The C170E and C221T mutations provided favorable interactions with glucooligosaccharide substrates at subsite +2, while the A219N mutation probably improved the substrate preference for cellobiose and gentiobiose relative to laminaribiose at subsite +1. The N407Y mutation increased the affinity toward cellooligosaccharides. These results give further insights into the molecular determinants responsible for substrate specificity in GH1 ß-glucosidases and may provide a basis for future enzyme engineering applications.

Tunable structure of chimeric isomaltomegalosaccharides with double α-(1 → 4)-glucosyl chains enhances the solubility of water-insoluble bioactive compounds.

Isomaltomegalosaccharides with α-(1 â†’ 4) and α-(1 â†’ 6)-segments solubilize water-insoluble ligands since the former complexes with the ligand and the latter solubilizes the complex. Previously, we enzymatically synthesized isomaltomegalosaccharide with a single α-(1 â†’ 4)-segment at the reducing end (S-IMS) by dextran dextrinase (DDase), but the chain length [average degree of polymerization (DP) ≤ 9] was insufficient for strong encapsulation. We hypothesized that the conjugation of longer α-(1 â†’ 4)-segment afforded the promising function although DDase is incapable to do so. In this study, the cyclodextrin glucanotransferase-catalyzed coupling reaction of α-cyclodextrin to S-IMS synthesized a new α-(1 â†’ 4)-segment at the nonreducing end (N-4S) of S-IMS to form D-IMS [IMS harboring double α-(1 â†’ 4)-segments]. The length of N-4S was modulated by the ratio between α-cyclodextrin and S-IMS, generating N-4Ss with DPs of 7-50. Based on phase-solubility analysis, D-IMS-28.3/13/3 bearing amylose-like helical N-4S with DP of 28.3 displayed a water-soluble complex with aromatic drugs and curcumin. Small-angle X-ray scattering revealed the chain adapted to rigid in solution in which the radius of gyration was estimated to 2.4 nm. Furthermore, D-IMS with short N-4S solubilized flavonoids of less-soluble multifunctional substances. In our research, enzyme-generated functional biomaterials from DDase were developed to maximize the hydrophobic binding efficacy towards water-insoluble bioactive compounds.

Crystal structure and identification of amino acid residues for catalysis and binding of GH3 AnBX ß-xylosidase from Aspergillus niger.

ß-Xylosidases catalyze the hydrolysis of xylooligosaccharides to xylose in the final step of hemicellulose degradation. AnBX, which is a GH3 ß-xylosidase from Aspergillus niger, has a high catalytic efficiency toward xyloside substrates. In this study, we report the three-dimensional structure and the identification of catalytic and substrate binding residues of AnBX by performing site-directed mutagenesis, kinetic analysis, and NMR spectroscopy-associated analysis of the azide rescue reaction. The structure of the E88A mutant of AnBX, determined at 2.5-Å resolution, contains two molecules in the asymmetric unit, each of which is composed of three domains, namely an N-terminal (ß/α)8 TIM-barrel-like domain, an (α/ß)6 sandwich domain, and a C-terminal fibronectin type III domain. Asp288 and Glu500 of AnBX were experimentally confirmed to act as the catalytic nucleophile and acid/base catalyst, respectively. The crystal structure revealed that Trp86, Glu88 and Cys289, which formed a disulfide bond with Cys321, were located at subsite -1. Although the E88D and C289W mutations reduced catalytic efficiency toward all four substrates tested, the substitution of Trp86 with Ala, Asp and Ser increased the substrate preference for glucoside relative to xyloside substrates, indicating that Trp86 is responsible for the xyloside specificity of AnBX. The structural and biochemical information of AnBX obtained in this study provides invaluable insight into modulating the enzymatic properties for the hydrolysis of lignocellulosic biomass. KEY POINTS: • Asp288 and Glu500 of AnBX are the nucleophile and acid/base catalyst, respectively • Glu88 and the Cys289-Cys321 disulfide bond are crucial for the catalytic activity of AnBX • The W86A and W86S mutations in AnBX increased the preference for glucoside substrates.

Nonreducing terminal chimeric isomaltomegalosaccharide and its integration with azoreductase for the remediation of soil-contaminated lipophilic azo dyes.

Lipophilic azo dyes are practically water-insoluble, and their dissolution by organic solvents and surfactants is harmful to biological treatment with living cells and enzymes. This study aimed to evaluate the feasibility of a newly synthesized nonreducing terminal chimeric isomaltomegalosaccharide (N-IMS) as a nontoxic solubilizer of four simulated lipophilic azo dye wastes for enzymatic degradation. N-IMS bearing a helical α-(1 â†’ 4)-glucosidic segment derived from a donor substrate α-cyclodextrin was produced by a coupling reaction of cyclodextrin glucanotransferase. Inclusion complexing by N-IMS overcame the solubility issue with equilibrium constants of 1786-242 M-1 (methyl yellow > ethyl red > methyl red > azo violet). Circular dichroism spectra revealed the axial alignment of the aromatic rings in the N-IMS cavity, while UV-visible absorption quenching revealed that the azo bond of methyl yellow was particularly induced. Desorption of the dyes from acidic and neutral soils was specific to aqueous organic over alkali extraction. The dissolution kinetics of the incorporated dyes followed a sigmoid pattern facilitating the subsequent decolorization process with azoreductase. It was demonstrated that after soil extraction, the solid dyes dissolved with N-IMS assistance and spontaneously digested by coupled azoreductase/glucose dehydrogenase (for a cofactor regeneration system) with the liberation of the corresponding aromatic amine.

Partial depolymerization of tamarind seed xyloglucan and its functionality toward enhancing the solubility of curcumin.

Polysaccharides of tamarind seed, a byproduct of the tamarind pulp industry, displayed a potential solubility improvement of lipophilic bioactive molecules but their textural characteristics hinder the dietary formulation. In contrast, the commonly available xyloglucan oligosaccharides (XOSs) with degrees of polymerization (DPs) of 7, 8, and 9 were too short to maintain their ability. The binding capacity of the between sizes is unknown due to a lack of appropriate preparation. We prepared xyloglucan megalosaccharides (XMSs) by partial depolymerization, where term megalosaccharide (MS) defines the middle chain-length saccharide between DPs 10 and 100. Digestion with fungal cellulase enabled reproducible active XMSs. Further identification of pure XMS segments indicated that XMS-B has an average DP of 17.2 (Gal3Glc8Xyl6) with a branched dimer of XOS 8 and 9 and was free of side-chain arabinose, the residue influencing high viscosity. Curcumin, a bioactive pigment, has poor bioavailability because of its water insolubility. XMSs with average DPs of 15.4-24.3 have similarly sufficient capacities to solubilize curcumin. The solubility of curcumin was improved 180-fold by the addition of 50 %, w/v, XMSs, which yielded a clear yellow liquid. Our findings indicated that XMSs were a promising added-value agent in foods and pharmaceuticals for the oral intake of curcumin.

Discovery of α-l-Glucosidase Raises the Possibility of α-l-Glucosides in Nature.

Glucose, a common monosaccharide in nature, is dominated by the d-enantiomer. Meanwhile, the discovery of l-glucose-utilizing bacteria and the elucidation of their metabolic pathways 10 years ago suggests that l-glucose exists naturally. Most carbohydrates exist as glycosides rather than monosaccharides; therefore, we expected that nature also contains l-glucosides. Sequence analysis within glycoside hydrolase family 29 led us to identify two α-l-glucosidases, ClAgl29A and ClAgl29B, derived from Cecembia lonarensis LW9. ClAgl29A and ClAgl29B exhibited higher K m, k cat, and k cat/K m values for p-nitrophenyl α-l-glucoside than that for p-nitrophenyl α-l-fucoside. Structural analysis of ClAgl29B in complex with l-glucose showed that these enzymes have an active-site pocket that preferentially binds α-l-glucoside, but excludes α-l-fucoside. These results suggest that ClAgl29A and ClAgl29B evolved to hydrolyze α-l-glucoside, implying the existence of α-l-glucoside in nature. Furthermore, α-l-glucosidic linkages (α-l-Glc-(1 → 3)-l-Glc, α-l-Glc-(1 → 2)-l-Glc, and α-l-Glc-(1 → 6)-l-Glc) were synthesized by the transglucosylation activity of ClAgl29A and ClAgl29B. We believe that this study will lead to new research on α-l-glucosides, including determining the physiological effects on humans, and the discovery of novel α-l-glucoside-related enzymes.

Physicochemical functionality of chimeric isomaltomegalosaccharides with α-(1 → 4)-glucosidic segments of various lengths.

Isomaltomegalosaccharide (IMS) is a long chimeric glucosaccharide composed of α-(1 â†’ 6)- and α-(1 â†’ 4)-linked segments at nonreducing and reducing ends, respectively; the hydrophilicity and hydrophobicity of these segments are expected to lead to bifunctionality. We enzymatically synthesized IMS with average degrees of polymerization (DPs) of 15.8, 19.3, and 23.5, where α-(1 â†’ 4)-segments had DPs of 3, 6, and 9, respectively. IMS exhibited considerably higher water solubility than maltodextrin because of the α-(1 â†’ 6)-segment and an identical resistance to thermal degradation as short dextran. Interaction of IMS with a fluorescent probe of 2-p-toluidinylnaphthalene-6-sulfonate demonstrated that IMS was more hydrophobic than maltodextrin, where the degree of hydrophobicity increased as DP of α-(1 â†’ 4)-segment increased (9 > 6 > 3). Fluorescent pyrene-estimating polarity of IMS was found to be similar to that of methanol or 1-butanol. The bifunctional IMS enhanced the water solubility of quercetin-3-O-glucoside and quercetin: the solubilization of less-soluble bioactive substances is beneficial in carbohydrate industry.

Characterization of an Unknown Region Linked to the Glycoside Hydrolase Family 17 ß-1,3-Glucanase of Vibrio vulnificus Reveals a Novel Glucan-Binding Domain.

The glycoside hydrolase family 17 ß-1,3-glucanase of Vibrio vulnificus (VvGH17) has two unknown regions in the N- and C-termini. Here, we characterized these domains by preparing mutant enzymes. VvGH17 demonstrated hydrolytic activity of ß-(1→3)-glucan, mainly producing laminaribiose, but not of ß-(1→3)/ß-(1→4)-glucan. The C-terminal-truncated mutants (ΔC466 and ΔC441) showed decreased activity, approximately one-third of that of the WT, and ΔC415 lost almost all activity. An analysis using affinity gel containing laminarin or barley ß-glucan revealed a shift in the mobility of the ΔC466, ΔC441, and ΔC415 mutants compared to the WT. Tryptophan residues showed a strong affinity for carbohydrates. Three of four point-mutations of the tryptophan in the C-terminus (W472A, W499A, and W542A) showed a reduction in binding ability to laminarin and barley ß-glucan. The C-terminus was predicted to have a ß-sandwich structure, and three tryptophan residues (Trp472, Trp499, and Trp542) constituted a putative substrate-binding cave. Linker and substrate-binding functions were assigned to the C-terminus. The N-terminal-truncated mutants also showed decreased activity. The WT formed a trimer, while the N-terminal truncations formed monomers, indicating that the N-terminus contributed to the multimeric form of VvGH17. The results of this study are useful for understanding the structure and the function of GH17 ß-1,3-glucanases.

A practical approach to producing isomaltomegalosaccharide using dextran dextrinase from Gluconobacter oxydans ATCC 11894.

Dextran dextrinase (DDase) catalyzes formation of the polysaccharide dextran from maltodextrin. During the synthesis of dextran, DDase also generates the beneficial material isomaltomegalosaccharide (IMS). The term megalosaccharide is used for a saccharide having DP = 10-100 or 10-200 (DP, degree of polymerization). IMS is a chimeric glucosaccharide comprising α-(1 → 6)- and α-(1 → 4)-linked portions at the nonreducing and reducing ends, respectively, in which the α-(1 → 4)-glucosyl portion originates from maltodextrin of the substrate. In this study, IMS was produced by a practical approach using extracellular DDase (DDext) or cell surface DDase (DDsur) of Gluconobacter oxydans ATCC 11894. DDsur was the original form, so we prepared DDext via secretion from intact cells by incubating with 0.5% G6/G7 (maltohexaose/maltoheptaose); this was followed by generation of IMS from various concentrations of G6/G7 substrate at different temperatures for 96 h. However, IMS synthesis by DDext was limited by insufficient formation of α-(1 → 6)-glucosidic linkages, suggesting that DDase also catalyzes elongation of α-(1 → 4)-glucosyl chain. For production of IMS using DDsur, intact cells bearing DDsur were directly incubated with 20% G6/G7 at 45 °C by optimizing conditions such as cell concentration and agitation efficiency, which resulted in generation of IMS (average DP = 14.7) with 61% α-(1 → 6)-glucosyl content in 51% yield. Increases in substrate concentration and agitation efficiency were found to decrease dextran formation and increase IMS production, which improved the reaction conditions for DDext. Under modified conditions (20% G6/G7, agitation speed of 100 rpm at 45 °C), DDext produced IMS (average DP = 14.5) with 65% α-(1 → 6)-glucosyl content in a good yield of 87%. KEY POINTS: • Beneficial IMS was produced using thermostabilized DDase. • Optimum conditions for reduced dextran formation were successfully determined. • A practical approach was established to provide IMS with a great yield of 87%.

Structural insights reveal the second base catalyst of isomaltose glucohydrolase.

Glycoside hydrolase family 15 (GH15) inverting enzymes contain two glutamate residues functioning as a general acid catalyst and a general base catalyst, for isomaltose glucohydrolase (IGHase), Glu178 and Glu335, respectively. Generally, a two-catalytic residue-mediated reaction exhibits a typical bell-shaped pH-activity curve. However, IGHase is found to display atypical non-bell-shaped pH-kcat and pH-kcat /Km profiles, theoretically better-fitted to a three-catalytic residue-associated pH-activity curve. We determined the crystal structure of IGHase by the single-wavelength anomalous dispersion method using sulfur atoms and the cocrystal structure of a catalytic base mutant E335A with isomaltose. Although the activity of E335A was undetectable, the electron density observed in its active site pocket did not correspond to an isomaltose but a glycerol and a ß-glucose, cryoprotectant, and hydrolysis product. Our structural and biochemical analyses of several mutant enzymes suggest that Tyr48 acts as a second catalytic base catalyst. Y48F mutant displayed almost equivalent specific activity to a catalytic acid mutant E178A. Tyr48, highly conserved in all GH15 members, is fixed by another Tyr residue in many GH15 enzymes; the latter Tyr is replaced by Phe290 in IGHase. The pH profile of F290Y mutant changed to a bell-shaped curve, suggesting that Phe290 is a key residue distinguishing Tyr48 of IGHase from other GH15 members. Furthermore, F290Y is found to accelerate the condensation of isomaltose from glucose by modifying a hydrogen-bonding network between Tyr290-Tyr48-Glu335. The present study indicates that the atypical Phe290 makes Tyr48 of IGHase unique among GH15 enzymes.

Molecular insight into regioselectivity of transfructosylation catalyzed by GH68 levansucrase and ß-fructofuranosidase.

Glycoside hydrolase family 68 (GH68) enzymes catalyze ß-fructosyltransfer from sucrose to another sucrose, the so-called transfructosylation. Although regioselectivity of transfructosylation is divergent in GH68 enzymes, there is insufficient information available on the structural factor(s) involved in the selectivity. Here, we found two GH68 enzymes, ß-fructofuranosidase (FFZm) and levansucrase (LSZm), encoded tandemly in the genome of Zymomonas mobilis, displayed different selectivity: FFZm catalyzed the ß-(2→1)-transfructosylation (1-TF), whereas LSZm did both of 1-TF and ß-(2→6)-transfructosylation (6-TF). We identified His79FFZm and Ala343FFZm and their corresponding Asn84LSZm and Ser345LSZm respectively as the structural factors for those regioselectivities. LSZm with the respective substitution of FFZm-type His and Ala for its Asn84LSZm and Ser345LSZm (N84H/S345A-LSZm) lost 6-TF and enhanced 1-TF. Conversely, the LSZm-type replacement of His79FFZm and Ala343FFZm in FFZm (H79N/A343S-FFZm) almost lost 1-TF and acquired 6-TF. H79N/A343S-FFZm exhibited the selectivity like LSZm but did not produce the ß-(2→6)-fructoside-linked levan and/or long levanooligosaccharides that LSZm did. We assumed Phe189LSZm to be a responsible residue for the elongation of levan chain in LSZm and mutated the corresponding Leu187FFZm in FFZm to Phe. An H79N/L187F/A343S-FFZm produced a higher quantity of long levanooligosaccharides than H79N/A343S-FFZm (or H79N-FFZm), although without levan formation, suggesting that LSZm has another structural factor for levan production. We also found that FFZm generated a sucrose analog, ß-D-fructofuranosyl α-D-mannopyranoside, by ß-fructosyltransfer to d-mannose and regarded His79FFZm and Ala343FFZm as key residues for this acceptor specificity. In summary, this study provides insight into the structural factors of regioselectivity and acceptor specificity in transfructosylation of GH68 enzymes.

Novel α-1,3/α-1,4-Glucosidase from Aspergillus niger Exhibits Unique Transglucosylation to Generate High Levels of Nigerose and Kojibiose.

α-Glucosidase from Aspergillus niger (AgdA; typical α-1,4-glucosidase) is known to industrially produce α-(1→6)-glucooligosaccharides. This fungus also has another α-glucosidase-like protein, AgdB. To learn its function, wild-type AgdB was expressed in Pichia pastoris. However, the enzyme displayed two electrophoretic forms due to heterogeneity of N-glycosylation at Asn354. The deglycosylation mutant N354D shared the same properties with wild-type AgdB. N354D demonstrated hydrolytic specificity toward α-(1→3)- and α-(1→4)-glucosidic linkages, indicating that AgdB is an α-1,3-/α-1,4-glucosidase. N354D-catalyzed transglucosylation from maltose was analyzed in short- and long-term reactions, enabling us to learn the transglucosylation specificity and product accumulation, respectively. A short-term reaction (<15 min) synthesized 3II- O-α-glucosyl-maltose and maltotriose, indicating α-1,3-/α-1,4-transferring specificity. A long-term reaction (<24 h) accumulated kojibiose and nigerose using formed glucose as an acceptor substrate. AgdA and AgdB are distinct α-glucosidases. At a high concentration of glucose added exogenously, AgdB largely generated the rare sugars kojibiose and nigerose (exhibiting beneficial physiological functions) with 19% and 24% yields from maltose, respectively.

Engineered dextranase from Streptococcus mutans enhances the production of longer isomaltooligosaccharides.

Herein, we investigated enzymatic properties and reaction specificities of Streptococcus mutans dextranase, which hydrolyzes α-(1→6)-glucosidic linkages in dextran to produce isomaltooligosaccharides. Reaction specificities of wild-type dextranase and its mutant derivatives were examined using dextran and a series of enzymatically prepared p-nitrophenyl α-isomaltooligosaccharides. In experiments with 4-mg·mL-1 dextran, isomaltooligosaccharides with degrees of polymerization (DP) of 3 and 4 were present at the beginning of the reaction, and glucose and isomaltose were produced by the end of the reaction. Increased concentrations of the substrate dextran (40 mg·mL-1) yielded isomaltooligosaccharides with higher DP, and the mutations T558H, W279A/T563N, and W279F/T563N at the -3 and -4 subsites affected hydrolytic activities of the enzyme, likely reflecting decreases in substrate affinity at the -4 subsite. In particular, T558H increased the proportion of isomaltooligosaccharide with DP of 5 in hydrolysates following reactions with 4-mg·mL-1 dextran.Abbreviations CI: cycloisomaltooligosaccharide; CITase: CI glucanotransferase; CITase-Bc: CITase from Bacillus circulans T-3040; DP: degree of polymerization of glucose unit; GH: glycoside hydrolase family; GTF: glucansucrase; HPAEC-PAD: high performance anion-exchange chromatography-pulsed amperometric detection; IG: isomaltooligosaccharide; IGn: IG with DP of n (n, 2‒5); PNP: p-nitrophenol; PNP-Glc: p-nitrophenyl α-glucoside; PNP-IG: p-nitrophenyl isomaltooligosaccharide; PNP-IGn: PNP-IG with DP of n (n, 2‒6); SmDex: dextranase from Streptococcus mutans; SmDexTM: S. mutans ATCC25175 SmDex bearing Gln100‒Ile732.

A novel glycoside hydrolase family 97 enzyme: Bifunctional ß-l-arabinopyranosidase/α-galactosidase from Bacteroides thetaiotaomicron.

Glycoside hydrolase family 97 (GH97) is one of the most interesting glycosidase families, which contains inverting and retaining glycosidases. Currently, only two enzyme types, α-glucoside hydrolase and α-galactosidase, are registered in the carbohydrate active enzyme database as GH97 function-known proteins. To explore new specificities, BT3661 and BT3664, which have distinct amino acid sequences when compared with that of GH97 α-glucoside hydrolase and α-galactosidase, were characterized in this study. BT3664 was identified to be an α-galactosidase, whereas BT3661 exhibits hydrolytic activity toward both ß-l-arabinopyranoside and α-d-galactopyranoside, and thus we designate BT3661 as a ß-l-arabinopyranosidase/α-d-galactosidase. Since this is the first dual substrate specificity enzyme in GH97, we investigated the substrate recognition mechanism of BT3661 by determining its three-dimensional structure and based on this structural data generated a number of mutants to probe the enzymatic mechanism. Structural comparison shows that the active-site pocket of BT3661 is similar to GH97 α-galactosidase BT1871, but the environment around the hydroxymethyl group of the galactopyranoside is different. While BT1871 bears Glu361 to stabilize the hydroxy group of C6 through a hydrogen bond with its carboxy group, BT3661 has Asn338 at the equivalent position. Amino acid mutation analysis indicates that the length of the side chain at Asn338 is important for defining specificity of BT3661. The kcat/Km value for the hydrolysis of p-nitrophenyl α-galactoside decreases when Asn338 is substituted with Glu, whereas an increase is observed when the mutation is Ala. Interestingly, mutation of Asn338 to Ala reduces the kcat/Km value for hydrolysis of p-nitrophenyl ß-l-arabinopyranoside.

Effects of mutation of Asn694 in Aspergillus niger α-glucosidase on hydrolysis and transglucosylation.

Aspergillus niger α-glucosidase (ANG), a member of glycoside hydrolase family 31, catalyzes hydrolysis of α-glucosidic linkages at the non-reducing end. In the presence of high concentrations of maltose, the enzyme also catalyzes the formation of α-(1→6)-glucosyl products by transglucosylation and it is used for production of the industrially useful panose and isomaltooligosaccharides. The initial transglucosylation by wild-type ANG in the presence of 100 mM maltose [Glc(α1-4)Glc] yields both α-(1→6)- and α-(1→4)-glucosidic linkages, the latter constituting ~25% of the total transfer reaction product. The maltotriose [Glc(α1-4)Glc(α1-4)Glc], α-(1→4)-glucosyl product disappears quickly, whereas the α-(1→6)-glucosyl products panose [Glc(α1-6)Glc(α1-4)Glc], isomaltose [Glc(α1-6)Glc], and isomaltotriose [Glc(α1-6)Glc(α1-6)Glc] accumulate. To modify the transglucosylation properties of ANG, residue Asn694, which was predicted to be involved in formation of the plus subsites of ANG, was replaced with Ala, Leu, Phe, and Trp. Except for N694A, the mutations enhanced the initial velocity of the α-(1→4)-transfer reaction to produce maltotriose, which was then degraded at a rate similar to that by wild-type ANG. With increasing reaction time, N694F and N694W mutations led to the accumulation of larger amounts of isomaltose and isomaltotriose than achieved with the wild-type enzyme. In the final stage of the reaction, the major product was panose (N694A and N694L) or isomaltose (N694F and N694W).

Substrate recognition of the catalytic α-subunit of glucosidase II from Schizosaccharomyces pombe.

The recombinant catalytic α-subunit of N-glycan processing glucosidase II from Schizosaccharomyces pombe (SpGIIα) was produced in Escherichia coli. The recombinant SpGIIα exhibited quite low stability, with a reduction in activity to <40% after 2-days preservation at 4 °C, but the presence of 10% (v/v) glycerol prevented this loss of activity. SpGIIα, a member of the glycoside hydrolase family 31 (GH31), displayed the typical substrate specificity of GH31 α-glucosidases. The enzyme hydrolyzed not only α-(1→3)- but also α-(1→2)-, α-(1→4)-, and α-(1→6)-glucosidic linkages, and p-nitrophenyl α-glucoside. SpGIIα displayed most catalytic properties of glucosidase II. Hydrolytic activity of the terminal α-glucosidic residue of Glc2Man3-Dansyl was faster than that of Glc1Man3-Dansyl. This catalytic α-subunit also removed terminal glucose residues from native N-glycans (Glc2Man9GlcNAc2 and Glc1Man9GlcNAc2) although the activity was low.

Efficient synthesis of α-galactosyl oligosaccharides using a mutant Bacteroides thetaiotaomicron retaining α-galactosidase (BtGH97b).

The preparation of a glycosynthase, a catalytic nucleophile mutant of a glycosidase, is a well-established strategy for the effective synthesis of glycosidic linkages. However, glycosynthases derived from α-glycosidases can give poor yields of desired products because they require generally unstable ß-glycosyl fluoride donors. Here, we investigate a transglycosylation catalyzed by a catalytic nucleophile mutant derived from a glycoside hydrolase family (GH) 97 α-galactosidase, using more stable ß-galactosyl azide and α-galactosyl fluoride donors. The mutant enzyme catalyzes the glycosynthase reaction using ß-galactosyl azide and α-galactosyl transfer from α-galactosyl fluoride with assistance of external anions. Formate was more effective at restoring transfer activity than azide. Kinetic analysis suggests that poor transglycosylation in the presence of the azide is because of low activity of the ternary complex between enzyme, ß-galactosyl azide and acceptor. A three-dimensional structure of the mutant enzyme in complex with the transglycosylation product, ß-lactosyl α-d-galactoside, was solved to elucidate the ligand-binding aspects of the α-galactosidase. Subtle differences at the ß→α loops 1, 2 and 3 of the catalytic TIM barrel of the α-galactosidase from those of a homologous GH97 α-glucoside hydrolase seem to be involved in substrate recognitions. In particular, the Trp residues in ß→α loop 1 have separate roles. Trp312 of the α-galactosidase appears to exclude the equatorial hydroxy group at C4 of glucosides, whereas the corresponding Trp residue in the α-glucoside hydrolase makes a hydrogen bond with this hydroxy group. The mechanism of α-galactoside recognition is conserved among GH27, 31, 36 and 97 α-galactosidases. DATABASE: The atomic coordinates (code: 5E1Q) have been deposited in the Protein Data Bank.

Functional characterization of UDP-rhamnose-dependent rhamnosyltransferase involved in anthocyanin modification, a key enzyme determining blue coloration in Lobelia erinus.

Because structural modifications of flavonoids are closely related to their properties, such as stability, solubility, flavor and coloration, characterizing the enzymes that catalyze the modification reactions can be useful for engineering agriculturally beneficial traits of flavonoids. In this work, we examined the enzymes involved in the modification pathway of highly glycosylated and acylated anthocyanins that accumulate in Lobelia erinus. Cultivar Aqua Blue (AB) of L. erinus is blue-flowered and accumulates delphinidin 3-O-p-coumaroylrutinoside-5-O-malonylglucoside-3'5'-O-dihydroxycinnamoylglucoside (lobelinins) in its petals. Cultivar Aqua Lavender (AL) is mauve-flowered, and LC-MS analyses showed that AL accumulated delphinidin 3-O-glucoside (Dp3G), which was not further modified toward lobelinins. A crude protein assay showed that modification processes of lobelinin were carried out in a specific order, and there was no difference between AB and AL in modification reactions after rhamnosylation of Dp3G, indicating that the lack of highly modified anthocyanins in AL resulted from a single mutation of rhamnosyltransferase catalyzing the rhamnosylation of Dp3G. We cloned rhamnosyltransferase genes (RTs) from AB and confirmed their UDP-rhamnose-dependent rhamnosyltransferase activities on Dp3G using recombinant proteins. In contrast, the RT gene in AL had a 5-bp nucleotide deletion, resulting in a truncated polypeptide without the plant secondary product glycosyltransferase box. In a complementation test, AL that was transformed with the RT gene from AB produced blue flowers. These results suggest that rhamnosylation is an essential process for lobelinin synthesis, and thus the expression of RT has a great impact on the flower color and is necessary for the blue color of Lobelia flowers.

Molecular insights into the mechanism of thermal stability of actinomycete mannanase.

Streptomyces thermolilacinus mannanase (StMan), which requires Ca(2+) for its enhanced thermal stability and hydrolysis activity, possesses two Ca(2+) -binding sites in loop6 and loop7. We evaluated the function of the Ca(2+) -binding site in loop7 and the hydrogen bond between residues Ser247 in loop6 and Asp279 in loop7. The Ca(2+) -binding in loop7 was involved only in thermal stability. Mutations of Ser247 or Asp279 retained the Ca(2+) -binding ability; however, mutants showed less thermal stability than StMan. Phylogenetic analysis indicated that most glycoside hydrolase family 5 subfamily 8 mannanases could be stabilized by Ca(2+) ; however, the mechanism of StMan thermal stability was found to be quite specific in some actinomycete mannanases.

Megalo-type α-1,6-glucosaccharides induce production of tumor necrosis factor α in primary macrophages via toll-like receptor 4 signaling.

The term "megalo-saccharide" is used for saccharides with ten or more saccharide units, whereas the term "oligo-saccharide" is used for saccharides containing fewer than ten monosaccharide units. Megalo-type α-1,6-glucosaccharide (M-IM) is a non-digestible saccharide and not utilized by intestinal bacteria, suggesting that ingested M-IM may encounter ileum Peyer's patches that contains immune cells such as macrophages. Macrophages are responsible for antigen incorporation and presentation during the initial step of immune responses. We investigated whether M-IMs modulate macrophage functions such as cytokine production, nitric oxide production, cell viability, and phagocytosis. Primary macrophages collected from the rats were cultured with the existence of M-IM or lipopolysaccharides (LPS). M-IM and LPS induced the production of tumor necrosis factor α (TNFα), interleukin 6 (IL6), and nitric oxide in the primary macrophages. The gene expression profile of inflammatory factors including TNFα, IL6, and ILlß in M-IM-stimulated cells was similar to that of LPS-stimulated cells. The M-IM did not affect phagocytosis in the primary macrophages. The M-IM-induced TNFα production was suppressed in the cells treated with a tolllike receptor 4 (TLR4) inhibitor called TAK-242. In conclusion, the M-IM modulates cytokine expression via TLR4 signaling and may play a role in the modulation of immune responses.