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

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

Comparisons of the amylolytic enzymes and malt starch hydrolysates of two barley cultivars, Hokudai 1 (the first cultivar developed in Japan) and Kitanohoshi (currently used cultivar for beer production).

Starch degradation in malted barley produces yeast-fermentable sugars. In this study, we compared the amylolytic enzymes and composition of the malt starch hydrolysates of two barley cultivars, Hokudai 1 (the first cultivar established in Japan) and Kitanohoshi (the currently used cultivar for beer production). Hokudai 1 malt contained lower activity of amylolytic enzymes than Kitanohoshi malt, although these cultivars contained α-amylase AMY2 and ß-amylase Bmy1 as the predominant enzymes. Malt starch hydrolysate of Hokudai 1 contained more limit dextrin and less yeast-fermentable sugars than that of Kitanohoshi. In mixed malt saccharification, a high Hokudai 1 malt ratio increased the limit dextrin levels and decreased the maltotriose and maltose levels. Even though Kitanohoshi malt contained more amylolytic enzymes than Hokudai 1 malt, addition of Kitanohoshi extract containing the amylolytic enzymes did not enhance malt starch degradation of Hokudai 1. Hokudai 1 malt starch was less degradable than Kitanohoshi malt starch.

Hydrolysis-transglycosylation of sucrose and production of ß-(2→1)-fructan by inulosucrase from Neobacillus drentensis 57N.

Inulin, ß-(2→1)-fructan, is a beneficial polysaccharide used as a functional food ingredient. Microbial inulosucrases (ISs), catalyzing ß-(2→1)-transfructosylation, produce ß-(2→1)-fructan from sucrose. In this study, we identified a new IS (NdIS) from the soil isolate, Neobacillus drentensis 57N. Sequence analysis revealed that, like other Bacillaceae ISs, NdIS consists of a glycoside hydrolase family 68 domain and shares most of the 1-kestose-binding residues of the archaeal IS, InuHj. Native and recombinant NdIS were characterized. NdIS is a homotetramer. It does not require calcium for activity. High performance liquid chromatography and 13C-nuclear magnetic resonance indicated that NdIS catalyzed the hydrolysis and ß-(2→1)-transfructosylation of sucrose to synthesize ß-(2→1)-fructan with chain lengths of 42 or more residues. The rate dependence on sucrose concentration followed hydrolysis-transglycosylation kinetics, and a 50% transglycosylation ratio was obtained at 344 m m sucrose. These results suggest that transfructosylation from sucrose to ß-(2→1)-fructan occurs predominantly to elongate the fructan chain because sucrose is an unfavorable acceptor.

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

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

Identification and characterization of extracellular GH3 ß-glucosidase from the pink snow mold fungus, Microdochium nivale.

Glycoside hydrolase family 3 (GH3) ß-glucosidase exists in many filamentous fungi. In phytopathogenic fungi, it is involved in fungal growth and pathogenicity. Microdochium nivale is a severe phytopathogenic fungus of grasses and cereals and is the causal agent of pink snow mold, but its ß-glucosidase has not been identified. In this study, a GH3 ß-glucosidase of M. nivale (MnBG3A) was identified and characterized. Among various p-nitrophenyl ß-glycosides, MnBG3A showed activity on d-glucoside (pNP-Glc) and slight activity on d-xyloside. In the pNP-Glc hydrolysis, substrate inhibition occurred (Kis = 1.6 m m), and d-glucose caused competitive inhibition (Ki = 0.5 m m). MnBG3A acted on ß-glucobioses with ß1-3, -6, -4, and -2 linkages, in descending order of kcat/Km. In contrast, the regioselectivity for newly formed products was limited to ß1-6 linkage. MnBG3A has similar features to those of ß-glucosidases from Aspergillus spp., but higher sensitivity to inhibitory effects.

Alteration of Substrate Specificity and Transglucosylation Activity of GH13_31 α-Glucosidase from Bacillus sp. AHU2216 through Site-Directed Mutagenesis of Asn258 on ß→α Loop 5.

α-Glucosidase catalyzes the hydrolysis of α-d-glucosides and transglucosylation. Bacillus sp. AHU2216 α-glucosidase (BspAG13_31A), belonging to the glycoside hydrolase family 13 subfamily 31, specifically cleaves α-(1→4)-glucosidic linkages and shows high disaccharide specificity. We showed previously that the maltose moiety of maltotriose (G3) and maltotetraose (G4), covering subsites +1 and +2 of BspAG13_31A, adopts a less stable conformation than the global minimum energy conformation. This unstable d-glucosyl conformation likely arises from steric hindrance by Asn258 on ß→α loop 5 of the catalytic (ß/α)8-barrel. In this study, Asn258 mutants of BspAG13_31A were enzymatically and structurally analyzed. N258G/P mutations significantly enhanced trisaccharide specificity. The N258P mutation also enhanced the activity toward sucrose and produced erlose from sucrose through transglucosylation. N258G showed a higher specificity to transglucosylation with p-nitrophenyl α-d-glucopyranoside and maltose than the wild type. E256Q/N258G and E258Q/N258P structures in complex with G3 revealed that the maltose moiety of G3 bound at subsites +1 and +2 adopted a relaxed conformation, whereas a less stable conformation was taken in E256Q. This structural difference suggests that stabilizing the G3 conformation enhances trisaccharide specificity. The E256Q/N258G-G3 complex formed an additional hydrogen bond between Met229 and the d-glucose residue of G3 in subsite +2, and this interaction may enhance transglucosylation.

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%.

Substrate specificity of glycoside hydrolase family 1 ß-glucosidase AtBGlu42 from Arabidopsis thaliana and its molecular mechanism.

Plants possess many glycoside hydrolase family 1 (GH1) ß-glucosidases, which physiologically function in cell wall metabolism and activation of bioactive substances, but most remain uncharacterized. One GH1 isoenzyme AtBGlu42 in Arabidopsis thaliana has been identified to hydrolyze scopolin using the gene deficient plants, but no enzymatic properties were obtained. Its sequence similarity to another functionally characterized enzyme Os1BGlu4 in rice suggests that AtBGlu42 also acts on oligosaccharides. Here, we show that the recombinant AtBGlu42 possesses high kcat/Km not only on scopolin, but also on various ß-glucosides, cellooligosaccharides, and laminarioligosaccharides. Of the cellooligosaccharides, cellotriose was the most preferred. The crystal structure, determined at 1.7 Å resolution, suggests that Arg342 gives unfavorable binding to cellooligosaccharides at subsite +3. The mutants R342Y and R342A showed the highest preference on cellotetraose or cellopentaose with increased affinities at subsite +3, indicating that the residues at this position have an important role for chain length specificity.

Enzymatic characteristics of D-mannose 2-epimerase, a new member of the acylglucosamine 2-epimerase superfamily.

Carbohydrate epimerases and isomerases are essential for the metabolism and synthesis of carbohydrates. In this study, Runella slithyformis Runsl_4512 and Dyadobacter fermentans Dfer_5652 were characterized from a cluster of uncharacterized proteins of the acylglucosamine 2-epimerase (AGE) superfamily. These proteins catalyzed the intramolecular conversion of D-mannose to D-glucose, whereas they did not act on ß-(1 → 4)-mannobiose, N-acetyl-D-glucosamine, and D-fructose, which are substrates of known AGE superfamily members. The kcat/Km values of Runsl_4512 and Dfer_5652 for D-mannose epimerization were 3.89 and 3.51 min-1 mM-1, respectively. Monitoring the Runsl_4512 reaction through 1H-NMR showed the formation of ß-D-glucose and ß-D-mannose from D-mannose and D-glucose, respectively. In the reaction with ß-D-glucose, ß-D-mannose was produced at the initial stage of the reaction, but not in the reaction with α-D-glucose. These results indicate that Runsl_4512 catalyzed the 2-epimerization of the ß-anomer substrate with a net retention of the anomeric configuration. Since 2H was obviously detected at the 2-C position of D-mannose and D-glucose in the equilibrated reaction mixture produced by Runsl_4512 in 2H2O, this enzyme abstracts 2-H from the substrate and adds another proton to the intermediate. This mechanism is in accordance with the mechanism proposed for the reactions of other epimerases of the AGE superfamily, that is, AGE and cellobiose 2-epimerase. Upon reaction with 500 g/L D-glucose at 50 °C and pH 8.0, Runsl_4512 and Dfer_5652 produced D-mannose with a 24.4 and 22.8% yield, respectively. These D-mannose yields are higher than those of other enzyme systems, and ME acts as an efficient biocatalyst for producing D-mannose.

Biochemical characteristics of maltose phosphorylase MalE from Bacillus sp. AHU2001 and chemoenzymatic synthesis of oligosaccharides by the enzyme.

Maltose phosphorylase (MP), a glycoside hydrolase family 65 enzyme, reversibly phosphorolyzes maltose. In this study, we characterized Bacillus sp. AHU2001 MP (MalE) that was produced in Escherichia coli. The enzyme exhibited phosphorolytic activity to maltose, but not to other α-linked glucobioses and maltotriose. The optimum pH and temperature of MalE for maltose-phosphorolysis were 8.1 and 45°C, respectively. MalE was stable at a pH range of 4.5-10.4 and at ≤40°C. The phosphorolysis of maltose by MalE obeyed the sequential Bi-Bi mechanism. In reverse phosphorolysis, MalE utilized d-glucose, 1,5-anhydro-d-glucitol, methyl α-d-glucoside, 2-deoxy-d-glucose, d-mannose, d-glucosamine, N-acetyl-d-glucosamine, kojibiose, 3-deoxy-d-glucose, d-allose, 6-deoxy-d-glucose, d-xylose, d-lyxose, l-fucose, and l-sorbose as acceptors. The kcat(app)/Km(app) value for d-glucosamine and 6-deoxy-d-glucose was comparable to that for d-glucose, and that for other acceptors was 0.23-12% of that for d-glucose. MalE synthesized α-(1→3)-glucosides through reverse phosphorolysis with 2-deoxy-d-glucose and l-sorbose, and synthesized α-(1→4)-glucosides in the reaction with other tested acceptors.

A Transposon Mutagenesis System for Bifidobacterium longum subsp. longum Based on an IS3 Family Insertion Sequence, ISBlo11.

Bifidobacteria are a major component of the intestinal microbiota in humans, particularly breast-fed infants. Therefore, elucidation of the mechanisms by which these bacteria colonize the intestine is desired. One approach is transposon mutagenesis, a technique currently attracting much attention because, in combination with next-generation sequencing, it enables exhaustive identification of genes that contribute to microbial fitness. We now describe a transposon mutagenesis system for Bifidobacterium longum subsp. longum 105-A (JCM 31944) based on ISBlo11, a native IS3 family insertion sequence. To build this system, xylose-inducible or constitutive bifidobacterial promoters were tested to drive the expression of full-length or a truncated form at the N terminus of the ISBlo11 transposase. An artificial transposon plasmid, pBFS12, in which ISBlo11 terminal inverted repeats are separated by a 3-bp spacer, was also constructed to mimic the transposition intermediate of IS3 elements. The introduction of this plasmid into a strain expressing transposase resulted in the insertion of the plasmid with an efficiency of >103 CFU/µg DNA. The plasmid targets random 3- to 4-bp sequences, but with a preference for noncoding regions. This mutagenesis system also worked at least in B. longum NCC2705. Characterization of a transposon insertion mutant revealed that a putative α-glucosidase mediates palatinose and trehalose assimilation, demonstrating the suitability of transposon mutagenesis for loss-of-function analysis. We anticipate that this approach will accelerate functional genomic studies of B. longum subsp. longumIMPORTANCE Several hundred species of bacteria colonize the mammalian intestine. However, the genes that enable such bacteria to colonize and thrive in the intestine remain largely unexplored. Transposon mutagenesis, combined with next-generation sequencing, is a promising tool to comprehensively identify these genes but has so far been applied only to a small number of intestinal bacterial species. In this study, a transposon mutagenesis system was established for Bifidobacterium longum subsp. longum, a representative health-promoting Bifidobacterium species. The system enables the identification of genes that promote colonization and survival in the intestine and should help illuminate the physiology of this species.

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).

α-Glucosidases and α-1,4-glucan lyases: structures, functions, and physiological actions.

α-Glucosidases (AGases) and α-1,4-glucan lyases (GLases) catalyze the degradation of α-glucosidic linkages at the non-reducing ends of substrates to release α-glucose and anhydrofructose, respectively. The AGases belong to glycoside hydrolase (GH) families 13 and 31, and the GLases belong to GH31 and share the same structural fold with GH31 AGases. GH13 and GH31 AGases show diverse functions upon the hydrolysis of substrates, having linkage specificities and size preferences, as well as upon transglucosylation, forming specific α-glucosidic linkages. The crystal structures of both enzymes were determined using free and ligand-bound forms, which enabled us to understand the important structural elements responsible for the diverse functions. A series of mutational approaches revealed features of the structural elements. In particular, amino-acid residues in plus subsites are of significance, because they regulate transglucosylation, which is used in the production of industrially valuable oligosaccharides. The recently solved three-dimensional structure of GLase from red seaweed revealed the amino-acid residues essential for lyase activity and the strict recognition of the α-(1 â†’ 4)-glucosidic substrate linkage. The former was introduced to the GH31 AGase, and the resultant mutant displayed GLase activity. GH13 and GH31 AGases hydrate anhydrofructose to produce glucose, suggesting that AGases are involved in the catabolic pathway used to salvage unutilized anhydrofructose.

Evaluation of acceptor selectivity of Lactococcus lactis ssp. lactis trehalose 6-phosphate phosphorylase in the reverse phosphorolysis and synthesis of a new sugar phosphate.

Trehalose 6-phosphate phosphorylase (TrePP), a member of glycoside hydrolase family 65, catalyzes the reversible phosphorolysis of trehalose 6-phosphate (Tre6P) with inversion of the anomeric configuration to produce ß-d-glucose 1-phosphate (ß-Glc1P) and d-glucose 6-phosphate (Glc6P). TrePP in Lactococcus lactis ssp. lactis (LlTrePP) is, alongside the phosphotransferase system, involved in the metabolism of trehalose. In this study, recombinant LlTrePP was produced and characterized. It showed its highest reverse phosphorolytic activity at pH 4.8 and 40°C, and was stable in the pH range 5.0-8.0 and at up to 30°C. Kinetic analyses indicated that reverse phosphorolysis of Tre6P proceeded through a sequential bi bi mechanism involving the formation of a ternary complex of the enzyme, ß-Glc1P, and Glc6P. Suitable acceptor substrates were Glc6P, and, at a low level, d-mannose 6-phosphate (Man6P). From ß-Glc1P and Man6P, a novel sugar phosphate, α-d-Glcp-(1↔1)-α-d-Manp6P, was synthesized with 51% yield.

Structural advantage of sugar beet α-glucosidase to stabilize the Michaelis complex with long-chain substrate.

The α-glucosidase from sugar beet (SBG) is an exo-type glycosidase. The enzyme has a pocket-shaped active site, but efficiently hydrolyzes longer maltooligosaccharides and soluble starch due to lower Km and higher kcat/Km for such substrates. To obtain structural insights into the mechanism governing its unique substrate specificity, a series of acarviosyl-maltooligosaccharides was employed for steady-state kinetic and structural analyses. The acarviosyl-maltooligosaccharides have a longer maltooligosaccharide moiety compared with the maltose moiety of acarbose, which is known to be the transition state analog of α-glycosidases. The clear correlation obtained between log Ki of the acarviosyl-maltooligosaccharides and log(Km/kcat) for hydrolysis of maltooligosaccharides suggests that the acarviosyl-maltooligosaccharides are transition state mimics. The crystal structure of the enzyme bound with acarviosyl-maltohexaose reveals that substrate binding at a distance from the active site is maintained largely by van der Waals interactions, with the four glucose residues at the reducing terminus of acarviosyl-maltohexaose retaining a left-handed single-helical conformation, as also observed in cycloamyloses and single helical V-amyloses. The kinetic behavior and structural features suggest that the subsite structure suitable for the stable conformation of amylose lowers the Km for long-chain substrates, which in turn is responsible for higher specificity of the longer substrates.

Kinetic properties and substrate inhibition of α-galactosidase from Aspergillus niger.

The recombinant AglB produced by Pichia pastoris exhibited substrate inhibition behavior for the hydrolysis of p-nitrophenyl α-galactoside, whereas it hydrolyzed the natural substrates, including galactomanno-oligosaccharides and raffinose family oligosaccharides, according to the Michaelian kinetics. These contrasting kinetic behaviors can be attributed to the difference in the dissociation constant of second substrate from the enzyme and/or to the ability of the leaving group of the substrates. The enzyme displays the grater kcat/Km values for hydrolysis of the branched α-galactoside in galactomanno-oligosaccharides than that of raffinose and stachyose. A sequence comparison suggested that AglB had a shallow active-site pocket, and it can allow to hydrolyze the branched α-galactosides, but not linear raffinose family oligosaccharides.

Purification and characterization of a chloride ion-dependent α-glucosidase from the midgut gland of Japanese scallop (Patinopecten yessoensis).

Marine glycoside hydrolases hold enormous potential due to their habitat-related characteristics such as salt tolerance, barophilicity, and cold tolerance. We purified an α-glucosidase (PYG) from the midgut gland of the Japanese scallop (Patinopecten yessoensis) and found that this enzyme has unique characteristics. The use of acarbose affinity chromatography during the purification was particularly effective, increasing the specific activity 570-fold. PYG is an interesting chloride ion-dependent enzyme. Chloride ion causes distinctive changes in its enzymatic properties, increasing its hydrolysis rate, changing the pH profile of its enzyme activity, shifting the range of its pH stability to the alkaline region, and raising its optimal temperature from 37 to 55 °C. Furthermore, chloride ion altered PYG's substrate specificity. PYG exhibited the highest Vmax/Km value toward maltooctaose in the absence of chloride ion and toward maltotriose in the presence of chloride ion.

Structural insights into the epimerization of ß-1,4-linked oligosaccharides catalyzed by cellobiose 2-epimerase, the sole enzyme epimerizing non-anomeric hydroxyl groups of unmodified sugars.

Cellobiose 2-epimerase (CE) reversibly converts d-glucose residues into d-mannose residues at the reducing end of unmodified ß1,4-linked oligosaccharides, including ß-1,4-mannobiose, cellobiose, and lactose. CE is responsible for conversion of ß1,4-mannobiose to 4-O-ß-d-mannosyl-d-glucose in mannan metabolism. However, the detailed catalytic mechanism of CE is unclear due to the lack of structural data in complex with ligands. We determined the crystal structures of halothermophile Rhodothermus marinus CE (RmCE) in complex with substrates/products or intermediate analogs, and its apo form. The structures in complex with the substrates/products indicated that the residues in the ß5-ß6 loop as well as those in the inner six helices form the catalytic site. Trp-322 and Trp-385 interact with reducing and non-reducing end parts of these ligands, respectively, by stacking interactions. The architecture of the catalytic site also provided insights into the mechanism of reversible epimerization. His-259 abstracts the H2 proton of the d-mannose residue at the reducing end, and consistently forms the cis-enediol intermediate by facilitated depolarization of the 2-OH group mediated by hydrogen bonding interaction with His-200. His-390 subsequently donates the proton to the C2 atom of the intermediate to form a d-glucose residue. The reverse reaction is mediated by these three histidines with the inverse roles of acid/base catalysts. The conformation of cellobiitol demonstrated that the deprotonation/reprotonation step is coupled with rotation of the C2-C3 bond of the open form of the ligand. Moreover, it is postulated that His-390 is closely related to ring opening/closure by transferring a proton between the O5 and O1 atoms of the ligand.

Structural analysis of the α-glucosidase HaG provides new insights into substrate specificity and catalytic mechanism.

α-Glucosidases, which catalyze the hydrolysis of the α-glucosidic linkage at the nonreducing end of the substrate, are important for the metabolism of α-glucosides. Halomonas sp. H11 α-glucosidase (HaG), belonging to glycoside hydrolase family 13 (GH13), only has high hydrolytic activity towards the α-(1 → 4)-linked disaccharide maltose among naturally occurring substrates. Although several three-dimensional structures of GH13 members have been solved, the disaccharide specificity and α-(1 → 4) recognition mechanism of α-glucosidase are unclear owing to a lack of corresponding substrate-bound structures. In this study, four crystal structures of HaG were solved: the apo form, the glucosyl-enzyme intermediate complex, the E271Q mutant in complex with its natural substrate maltose and a complex of the D202N mutant with D-glucose and glycerol. These structures explicitly provide insights into the substrate specificity and catalytic mechanism of HaG. A peculiar long ß â†’ α loop 4 which exists in α-glucosidase is responsible for the strict recognition of disaccharides owing to steric hindrance. Two residues, Thr203 and Phe297, assisted with Gly228, were found to determine the glycosidic linkage specificity of the substrate at subsite +1. Furthermore, an explanation of the α-glucosidase reaction mechanism is proposed based on the glucosyl-enzyme intermediate structure.

Extracellular and cell-associated forms of Gluconobacter oxydans dextran dextrinase change their localization depending on the cell growth.

Gluconobacter oxydans ATCC 11894 produces dextran dextrinase (DDase, EC 2.4.1.2), which synthesizes dextran from the starch hydrolysate, dextrin and is known to cause ropy beer. G. oxydans ATCC 11894 was believed to possess both a secreted DDase (DDext) and an intracellular DDase (DDint), expressed upon cultivation with dextrin and glucose, respectively. However, genomic Southern blot, peptide mass fingerprinting and reaction product-pattern analyses revealed that both DDext and DDint were identical. The activity in the cell suspension and its liberation from the spheroplast cells indicated that DDint was localized on the cell surface. The localization of DDase was altered during the culture depending on the growth phase. During the early growth stage, DDase was exclusively liberated into the medium (DDext), and the cell-associated form (DDint) appeared after depletion of glucose from the medium.