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.

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.

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%.

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.

Polyglycolic acid mesh for preventing post-thoracoscopic bullectomy recurrence.

PURPOSE: Thoracoscopic bullectomy is a common treatment modality for spontaneous pneumothorax but can result in a high frequency of postoperative recurrent pneumothorax in young patients. This retrospective study compared the recurrence rate of pneumothorax following conventional thoracoscopic bullectomy to that following bullectomy using a low-density polyglycolic acid mesh to cover the staple line. METHODS: Group A comprised 237 patients who experienced 294 episodes of pneumothorax and underwent thoracoscopic bullectomy alone, and Group B comprised 130 patients who experienced 155 episodes of pneumothorax and underwent bullectomy with polyglycolic acid mesh used to cover the visceral pleura. To compare the postoperative inflammatory response between the two groups, we measured three inflammatory parameters: highest body temperature after surgery, C-reactive protein level on postoperative day 3, and change in eosinophil count from the day before the surgery to postoperative day 3. RESULTS: The recurrence rate was significantly lower in Group B than in Group A (2.6% vs. 24.8%, P < 0.000001). All three inflammatory parameters were significantly higher in Group B than in Group A. CONCLUSIONS: Using a polyglycolic acid mesh covering after thoracoscopic bullectomy resulted in acceptable long-term results (recurrence rate: 2.6%). This method was associated with a slightly elevated inflammatory response.

A novel intracellular dextranase derived from Paenibacillus sp. 598K with an ability to degrade cycloisomaltooligosaccharides.

Paenibacillus sp. 598K produces cycloisomaltooligosaccharides (CIs) in culture from dextran and starch. CIs are cyclic oligosaccharides consisting of seven or more α-(1 → 6)-linked-D-glucose residues. The extracellular enzyme CI glucanotransferase (PsCITase), which is the member of glycoside hydrolase family 66, catalyzes the final stage of CI production and produces mainly cycloisomaltoheptaose. We have discovered a novel intracellular CI-degrading dextranase (PsDEX598) from Paenibacillus sp. 598K. The 69.7-kDa recombinant PsDEX598 does not digest isomaltotetraose or shorter isomaltooligosaccharides, but digests longer ones of at least up to isomaltoheptaose. It also digests oligoCIs of cycloisomaltoheptaose, cycloisomaltooctaose, and cycloisomaltononaose better than it does with megaloCIs of cycloisomaltodecaose, cycloisomaltoundecaose, and cycloisomaltododecaose, as well as an α-(1 → 6)-D-glucan of dextran 40. PsDEX598 is produced intracellularly when culture medium is supplemented with cycloisomaltoheptaose or dextran, but not with isomaltooligosaccharides (a mixture of isomaltose, isomaltotriose, and panose), starch, or glucose. The whole genomic DNA sequence of the strain 598K implies that it harbors two genes for enzymes belonging to glycoside hydrolase family 66 (PsCITase and PsDEX598), and PsDEX598 is the only dextranase in the strain. PsDEX598 does not have any carbohydrate-binding modules (CBMs) and has a low similarity (< 30%) with other family 66 dextranases, and the catalytic amino acids of this enzyme are predicted to be Asp191, Asp303, and Glu368. The strain Paenibacillus sp. 598K appears to take up CI-7, so these findings indicate that this bacterium can degrade CIs using a dextranase within the cells and so utilize them as a carbon source for growth.

Enzymatically synthesized megalo-type isomaltosaccharides enhance the barrier function of the tight junction in the intestinal epithelium.

Megalo-type isomaltosaccharides are an enzymatically synthesized foodstuff produced by transglucosylation from maltodextrin, and they contain a mid-chain length polymer of D-glucose with α-1,6-glycoside linkages. The injection of a solution of megalo-type isomaltosaccharides (1-4%(w/v), average DP = 12.6), but not oligo-type isomaltosaccharides (average DP = 3.3), into the intestinal lumen dose-dependently reduced the transport rates of tight junction permeable markers in a ligated loop of the anesthetized rat jejunum. Application of the megalosaccharide also suppressed the transport of tight junction markers and enhanced transepithelial electrical resistance (TEER) in Caco-2 cell monolayers. Cholesterol sequestration by methyl-ß-cyclodextrin in the Caco-2 monolayers abolished the effect of megalosaccharide. Treatment with anti-caveolin-1 and a caveolae inhibitor, but not clathrin-dependent endocytosis and macropinocytosis inhibitors, suppressed the increase in TEER. These results indicate that isomaltosaccharides promote the barrier function of tight junctions in the intestinal epithelium in a chain-length dependent manner and that caveolae play a role in the effect.

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.

Carbohydrate-binding architecture of the multi-modular α-1,6-glucosyltransferase from Paenibacillus sp. 598K, which produces α-1,6-glucosyl-α-glucosaccharides from starch.

Paenibacillus sp. 598K α-1,6-glucosyltransferase (Ps6TG31A), a member of glycoside hydrolase family 31, catalyzes exo-α-glucohydrolysis and transglucosylation and produces α-1,6-glucosyl-α-glucosaccharides from α-glucan via its disproportionation activity. The crystal structure of Ps6TG31A was determined by an anomalous dispersion method using a terbium derivative. The monomeric Ps6TG31A consisted of one catalytic (ß/α)8-barrel domain and six small domains, one on the N-terminal and five on the C-terminal side. The structures of the enzyme complexed with maltohexaose, isomaltohexaose, and acarbose demonstrated that the ligands were observed in the catalytic cleft and the sugar-binding sites of four ß-domains. The catalytic site was structured by a glucose-binding pocket and an aglycon-binding cleft built by two sidewalls. The bound acarbose was located with its non-reducing end pseudosugar docked in the pocket, and the other moieties along one sidewall serving three subsites for the α-1,4-glucan. The bound isomaltooligosaccharide was found on the opposite sidewall, which provided the space for the acceptor molecule to be positioned for attack of the catalytic intermediate covalent complex during transglucosylation. The N-terminal domain recognized the α-1,4-glucan in a surface-binding mode. Two of the five C-terminal domains belong to the carbohydrate-binding modules family 35 and one to family 61. The sugar complex structures indicated that the first family 35 module preferred α-1,6-glucan, whereas the second family 35 module and family 61 module preferred α-1,4-glucan. Ps6TG31A appears to have enhanced transglucosylation activity facilitated by its carbohydrate-binding modules and substrate-binding cleft that positions the substrate and acceptor sugar for the transglucosylation.

Two Novel Glycoside Hydrolases Responsible for the Catabolism of Cyclobis-(1→6)-α-nigerosyl.

The actinobacterium Kribbella flavida NBRC 14399(T) produces cyclobis-(1→6)-α-nigerosyl (CNN), a cyclic glucotetraose with alternate α-(1→6)- and α-(1→3)-glucosidic linkages, from starch in the culture medium. We identified gene clusters associated with the production and intracellular catabolism of CNN in the K. flavida genome. One cluster encodes 6-α-glucosyltransferase and 3-α-isomaltosyltransferase, which are known to coproduce CNN from starch. The other cluster contains four genes annotated as a transcriptional regulator, sugar transporter, glycoside hydrolase family (GH) 31 protein (Kfla1895), and GH15 protein (Kfla1896). Kfla1895 hydrolyzed the α-(1→3)-glucosidic linkages of CNN and produced isomaltose via a possible linear tetrasaccharide. The initial rate of hydrolysis of CNN (11.6 s(-1)) was much higher than that of panose (0.242 s(-1)), and hydrolysis of isomaltotriose and nigerose was extremely low. Because Kfla1895 has a strong preference for the α-(1→3)-isomaltosyl moiety and effectively hydrolyzes the α-(1→3)-glucosidic linkage, it should be termed 1,3-α-isomaltosidase. Kfla1896 effectively hydrolyzed isomaltose with liberation of ß-glucose, but displayed low or no activity toward CNN and the general GH15 enzyme substrates such as maltose, soluble starch, or dextran. The kcat/Km for isomaltose (4.81 ± 0.18 s(-1) mm(-1)) was 6.9- and 19-fold higher than those for panose and isomaltotriose, respectively. These results indicate that Kfla1896 is a new GH15 enzyme with high substrate specificity for isomaltose, suggesting the enzyme should be designated an isomaltose glucohydrolase. This is the first report to identify a starch-utilization pathway that proceeds via CNN.

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).

Paenibacillus sp. 598K 6-α-glucosyltransferase is essential for cycloisomaltooligosaccharide synthesis from α-(1 → 4)-glucan.

Paenibacillus sp. 598K produces cycloisomaltooligosaccharides (cyclodextrans) from starch even in the absence of dextran. Cycloisomaltooligosaccharide glucanotransferase synthesizes cycloisomaltooligosaccharides exclusively from an α-(1 â†’ 6)-consecutive glucose chain consisting of at least four molecules. Starch is not a substrate of this enzyme. Therefore, we predicted that the bacterium possesses another enzyme system for extending α-(1 â†’ 6)-linked glucoses from starch, which can be used as the substrate for cycloisomaltooligosaccharide glucanotransferase, and identified the transglucosylation enzyme Ps6GT31A. We purified Ps6GT31A from the bacterial culture supernatant, cloned its corresponding gene, and characterized the recombinant enzyme. Ps6GT31A belongs to glycoside hydrolase family 31, and it liberates glucose from the non-reducing end of the substrate in the following order of activity: α-(1 â†’ 4)-> α-(1 â†’ 2)- > α-(1 â†’ 3)- > α-(1 â†’ 6)-glucobiose and maltopentaose > maltotetraose > maltotriose > maltose. Ps6GT31A catalyzes both hydrolysis and transglucosylation. The resulting transglucosylation compounds were analyzed by high-performance liquid chromatography and mass spectrometry. Analysis of the initial products by 13C nuclear magnetic resonance spectroscopy revealed that Ps6GT31A had a strong α-(1 â†’ 4) to α-(1 â†’ 6) transglucosylation activity. Ps6GT31A elongated α-(1 â†’ 6)-linked glucooligosaccharide to at least a degree of polymerization of 10 through a successive transglucosylation reaction. Eventually, cycloisomaltooligosaccharide glucanotransferase creates cycloisomaltooligosaccharides using the transglucosylation products generated by Ps6GT31A as the substrates. Our data suggest that Ps6GT31A is the key enzyme to synthesize α-(1 â†’ 6)-glucan for cycloisomaltooligosaccharide production in dextran-free environments.

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.

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.

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.

Molecular engineering of cycloisomaltooligosaccharide glucanotransferase from Bacillus circulans T-3040: structural determinants for the reaction product size and reactivity.

Cycloisomaltooligosaccharide glucanotransferase (CITase) is a member of glycoside hydrolase family 66 and it produces cycloisomaltooligosaccharides (CIs). Small CIs (CI-7-9) and large CIs (CI-≥10) are designated as oligosaccharide-type CIs (oligo-CIs) and megalosaccharide-type CIs (megalo-CIs) respectively. CITase from Bacillus circulans T-3040 (BcCITase) produces mainly CI-8 with little megalo-CIs. It has two family 35 carbohydrate-binding modules (BcCBM35-1 and BcCBM35-2). BcCBM35-1 is inserted in a catalytic domain of BcCITase and BcCBM35-2 is located at the C-terminal region. Our previous studies suggested that BcCBM35-1 has two substrate-binding sites (B-1 and B-2) [Suzuki et al. (2014) J. Biol. Chem. 289, 12040-12051]. We implemented site-directed mutagenesis of BcCITase to explore the preference for product size on the basis of the 3D structure of BcCITase. Mutational studies provided evidence that B-1 and B-2 contribute to recruiting substrate and maintaining product size respectively. A mutant (mutant-R) with four mutations (F268V, D469Y, A513V and Y515S) produced three times as much megalo-CIs (CI-10-12) and 1.5 times as much total CIs (CI-7-12) as compared with the wild-type (WT) BcCITase. The 3D structure of the substrate-enzyme complex of mutant-R suggested that the modified product size specificity was attributable to the construction of novel substrate-binding sites in the B-2 site of BcCBM35-1 and reactivity was improved by mutation on subsite -3 on the catalytic domain.

Structural elucidation of the cyclization mechanism of α-1,6-glucan by Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase.

Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase belongs to the glycoside hydrolase family 66 and catalyzes an intramolecular transglucosylation reaction that produces cycloisomaltooligosaccharides from dextran. The crystal structure of the core fragment from Ser-39 to Met-738 of B. circulans T-3040 cycloisomaltooligosaccharide glucanotransferase, devoid of its N-terminal signal peptide and C-terminal nonconserved regions, was determined. The structural model contained one catalytic (ß/α)8-barrel domain and three ß-domains. Domain N with an immunoglobulin-like ß-sandwich fold was attached to the N terminus; domain C with a Greek key ß-sandwich fold was located at the C terminus, and a carbohydrate-binding module family 35 (CBM35) ß-jellyroll domain B was inserted between the 7th ß-strand and the 7th α-helix of the catalytic domain A. The structures of the inactive catalytic nucleophile mutant enzyme complexed with isomaltohexaose, isomaltoheptaose, isomaltooctaose, and cycloisomaltooctaose revealed that the ligands bound in the catalytic cleft and the sugar-binding site of CBM35. Of these, isomaltooctaose bound in the catalytic site extended to the second sugar-binding site of CBM35, which acted as subsite -8, representing the enzyme·substrate complex when the enzyme produces cycloisomaltooctaose. The isomaltoheptaose and cycloisomaltooctaose bound in the catalytic cleft with a circular structure around Met-310, representing the enzyme·product complex. These structures collectively indicated that CBM35 functions in determining the size of the product, causing the predominant production of cycloisomaltooctaose by the enzyme. The canonical sugar-binding site of CBM35 bound the mid-part of isomaltooligosaccharides, indicating that the original function involved substrate binding required for efficient catalysis.

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.