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)-alpha-nigerosyl (CNN), a cyclic glucotetraose with alternate alpha-(1 -> 6)- and alpha-(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-alpha-glucosyl-transferase and 3-alpha-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 alpha-(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 alpha-(1 -> 3)-isomaltosyl moiety and effectively hydrolyzes the alpha-(1 -> 3)-glucosidic linkage, it should be termed 1,3-alpha-isomaltosidase. Kfla1896 effectively hydrolyzed isomaltose with liberation of beta-glucose, but displayed low or no activity toward CNN and the general GH15 enzyme substrates such as maltose, soluble starch, or dextran. The k(cat)/K-m 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

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

Isomaltomegalosaccharide (IMS) is a long chimeric glucosaccharide composed of alpha-(1-+ 6)-and alpha-(1-+ 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 alpha-(1-+ 4)-segments had DPs of 3, 6, and 9, respectively. IMS exhibited considerably higher water solubility than maltodextrin because of the alpha-(1-+ 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 alpha-(1-+ 4)-segment increased (9 > 6 > 3). Fluorescent pyreneestimating 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

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^) was much higher than that of panose (0.242 s^), 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 β-glucose, but displayed low or no activity toward CNN and the general GH15 enzyme substrates such as maltose, soluble starch, or dextran. The k_/K_m for isomaltose (4.81 ± 0.18^ mm^) 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

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-k(cat) and pH-k(cat)/K-m 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 beta-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

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 alpha-(1 -> 6)- and alpha-(1 -> 4)-linked portions at the nonreducing and reducing ends, respectively, in which the alpha-(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 alpha-(1 -> 6)-glucosidic linkages, suggesting that DDase also catalyzes elongation of alpha-(1 -> 4)-glucosyl chain. For production of IMS using DDsur, intact cells bearing DDsur were directly incubated with 20% G6/G7 at 45 degrees C by optimizing conditions such as cell concentration and agitation efficiency, which resulted in generation of IMS (average DP =14.7) with 61% alpha-(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 degrees C), DDext produced IMS (average DP =14.5) with 65% alpha-(1 -> 6)-glucosyl content in a good yield of 87%

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

Glycoside hydrolase family 68 (GH68) enzymes catalyze beta-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, beta-fructofuranosidase (FFZm) and levansucrase (LSZm), encoded tandemly in the genome of Zymomonas mobilis, displayed different selectivity: FFZm catalyzed the beta-(2 -> 1)-trans-fructosylation (1-TF), whereas LSZm did both of 1-TF and beta-(2 -> 6)-trans-fructosylation (6-TF). We identified His79(FFZm) and Ala343(FFZm) and their corresponding Asn84(LSZm) and Ser345(LSZm) respectively as the structural factors for those regioselectivities. LSZm with the respective substitution of FFZm-type His and Ala for its Asn84(LSZm) and Ser345(LSZm) (N84H/S345A-LSZm) lost 6-TF and enhanced 1-TF. Conversely, the LSZm-type replacement of His79(FFZm) and Ala343(FFZm) 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 beta-(2 -> 6)-fructoside-linked levan and/or long levanooligo-saccharides that LSZm did. We assumed Phe189(LSZm) to be a responsible residue for the elongation of levan chain in LSZm and mutated the corresponding Leu187(FFZm) 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, beta-D-fructofuranosyl alpha-D-mannopyranoside, by beta-fructosyltransfer to D-mannose and regarded His79(FFZm) and Ala343(FFZm) 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