Isomaltohexaose

Structural identification of carbohydrate isomers using ambient infrared-assisted dissociation.[Pubmed:37230717]

Anal Chim Acta. 2023 Jul 11;1264:341307.

Informative dissociation of carbohydrates using an infrared (IR) irradiation system is demonstrated under ambient conditions without the instrumentation of a mass spectrometer. Structural identification of carbohydrates and associated conjugates is essential for understanding their biological functions, but identification remains challenging. Herein, an easy and rugged method is reported for the structural identification of model carbohydrates, including Globo-H, three trisaccharide isomers (nigerotriose/laminaritriose/cellotriose), and two hexasaccharide isomers (laminarihexaose/Isomaltohexaose). For Globo-H, the numbers of cross-ring cleavages increased by factors of 4.4 and 3.4 upon ambient IR exposure, compared to an untreated control and a collision-induced dissociation (CID) sample. Moreover, 25-82% enhancement in the numbers of glycosidic bond cleavages upon ambient IR exposure was also obtained compared to untreated and CID samples. Unique features of first-generation fragments produced by ambient IR facilitated the differentiation of three trisaccharide isomers. Semi-quantitative analysis was achieved (coefficient of determination (R(2)) of 0.982) in a mixture of two hexasaccharide isomers via unique features generated upon ambient IR. Photothermal and radical migration effects induced by ambient IR were postulated as responsible for promoting carbohydrate fragmentation. This easy and rugged method could be a universally applicable protocol and complementary to other techniques for detailed structural characterization of carbohydrates.

Characterization of an Alkaline GH49 Dextranase from Marine Bacterium Arthrobacter oxydans KQ11 and Its Application in the Preparation of Isomalto-Oligosaccharide.[Pubmed:31430863]

Mar Drugs. 2019 Aug 19;17(8):479.

A GH49 dextranase gene DexKQ was cloned from marine bacteria Arthrobacter oxydans KQ11. It was recombinantly expressed using an Escherichia coli system. Recombinant DexKQ dextranase of 66 kDa exhibited the highest catalytic activity at pH 9.0 and 55 degrees C. kcat/Km of recombinant DexKQ at the optimum condition reached 3.03 s(-1) muM(-1), which was six times that of commercial dextranase (0.5 s(-1) muM(-1)). DexKQ possessed a Km value of 67.99 microM against dextran T70 substrate with 70 kDa molecular weight. Thin-layer chromatography (TLC) analysis showed that main hydrolysis end products were isomalto-oligosaccharide (IMO) including isomaltotetraose, isomaltopantose, and Isomaltohexaose. When compared with glucose, IMO could significantly improve growth of Bifidobacterium longum and Lactobacillus rhamnosus and inhibit growth of Escherichia coli and Staphylococcus aureus. This is the first report of dextranase from marine bacteria concerning recombinant expression and application in isomalto-oligosaccharide preparation.

Carbohydrate-binding architecture of the multi-modular alpha-1,6-glucosyltransferase from Paenibacillus sp. 598K, which produces alpha-1,6-glucosyl-alpha-glucosaccharides from starch.[Pubmed:28698247]

Biochem J. 2017 Aug 7;474(16):2763-2778.

Paenibacillus sp. 598K alpha-1,6-glucosyltransferase (Ps6TG31A), a member of glycoside hydrolase family 31, catalyzes exo-alpha-glucohydrolysis and transglucosylation and produces alpha-1,6-glucosyl-alpha-glucosaccharides from alpha-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 (beta/alpha)(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 beta-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 alpha-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 alpha-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 alpha-1,6-glucan, whereas the second family 35 module and family 61 module preferred alpha-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.

Crystal structure of thermophilic dextranase from Thermoanaerobacter pseudethanolicus.[Pubmed:26494689]

J Biochem. 2016 Mar;159(3):331-9.

The crystal structures of the wild type and catalytic mutant Asp-312-->Gly in complex with Isomaltohexaose of endo-1,6-dextranase from the thermophilic bacterium Thermoanaerobacter pseudethanolicus (TpDex), belonging to the glycoside hydrolase family 66, were determined. TpDex consists of three structural domains, a catalytic domain comprising an (beta/alpha)8-barrel and two beta-domains located at both N- and C-terminal ends. The Isomaltohexaose-complex structure demonstrated that the Isomaltohexaose molecule was bound across the catalytic site, showing that TpDex had six subsites (-4 to +2) in the catalytic cleft. Marked movement of the Trp-376 side-chain along with loop 6, which was the side wall component of the cleft at subsite +1, was observed to occupy subsite +1, indicating that it might expel the cleaved aglycone subsite after the hydrolysis reaction. Structural comparison with other mesophilic enzymes indicated that several structural features of TpDex, loop deletion, salt bridge and surface-exposed charged residue, may contribute to thermostability.

Structural elucidation of the cyclization mechanism of alpha-1,6-glucan by Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase.[Pubmed:24616103]

J Biol Chem. 2014 Apr 25;289(17):12040-12051.

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 (beta/alpha)8-barrel domain and three beta-domains. Domain N with an immunoglobulin-like beta-sandwich fold was attached to the N terminus; domain C with a Greek key beta-sandwich fold was located at the C terminus, and a carbohydrate-binding module family 35 (CBM35) beta-jellyroll domain B was inserted between the 7th beta-strand and the 7th alpha-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.

Reducing values: dinitrosalicylate gives over-oxidation and invalid results whereas copper bicinchoninate gives no over-oxidation and valid results.[Pubmed:24021436]

Carbohydr Res. 2013 Oct 18;380:118-23.

A comparative study was made between two carbohydrate reducing value methods, a relatively old, highly alkaline, 3,5-dinitrosalicylic acid (DNSA) method and a relatively newer, low alkaline (pH 10.5), copper bicinchoninate (CuBic) method. Reducing values for a series of equimolar amounts of maltose-maltohexaose, isomaltose-Isomaltohexaose, and cellobiose-cellohexaose were compared by the two methods. The DNSA method gave over-oxidation for equimolar amounts of all three of the oligosaccharide series. The amount of oxidation increased as the sizes of the oligosaccharides increased, giving inflated, inaccurate reducing values. The CuBic method gave constant reducing values, for equimolar amounts of the oligosaccharides, indicating that there was no over-oxidation, as the sizes of the oligosaccharides were increased. The two methods were used to determine the number average molecular weights (MWn) for six polysaccharides. The DNSA method was not able to determine the MWn for any of the polysaccharides tested due to the low sensitivity of the method, compared with the CuBic method that did not give over-oxidation and gave reasonable MWn values for all six of the polysaccharides tested.

Search for a dextransucrase minimal motif involved in dextran binding.[Pubmed:17826770]

FEBS Lett. 2007 Oct 2;581(24):4675-80.

Fourteen truncated forms of Leuconostoc mesenteroides NRRL B512-F dextransucrase, involving N-, C- or N- plus C-terminal domain truncations were tested for their ability to bind dextrans. The shortest fragment (14kDa molecular weight) that still exhibited a strong interaction with dextran was localized between amino acids N1397 and A1527 of the C-terminal domain (GBD-7) and consists of six YG repeats. With a dissociation constant K(d) of 2.8x10(-9)M, this motif shows a very high affinity for Isomaltohexaose and longer dextrans, supporting the proposed role of GBD in polymer formation. The potential application of GBD-7 as an affinity tag onto cheap resins like Sephacryl S300HR for rapid purification was evaluated and is discussed.

Subsite mapping of Aspergillus niger glucoamylases I and II with malto- and isomaltooligosaccharides.[Pubmed:18588152]

Biotechnol Bioeng. 1989 Aug 20;34(5):681-8.

Glucoamylase, industrially derived from Aspergillus niger, was chromatographically separated into forms I and II and purified to near homogeneity. Preparations were proved to be free of D-glucosyltransferase by electrophoretic and differential inhibition tests. Maximum rates and Michaelis constants were obtained for both glucoamylases I and II with maltooligosaccharides from maltose to maltoheptaose and with isomaltooligosaccharides from isomaltose to Isomaltohexaose. Subsite maps were calculated from these kinetic data and were not significantly different for the two forms. Subsites in both forms had lower affinities for D-glucosyl residues contained in isomaltooligosaccharides than for D-glucosyl residues in maltooligosaccharides.

Variable region cDNA sequences and antigen binding specificity of mouse monoclonal antibodies to isomaltosyl oligosaccharides coupled to proteins. T-dependent analogues of alpha(1----6)dextran.[Pubmed:2464028]

J Immunol. 1989 Feb 1;142(3):863-70.

Four mouse hybridomas specific for alpha(1----6)dextran, 16.4.12E (IgA kappa, C57BL/6), 28.4.10A (IgM kappa, BALB/c), 35.8.2H (IgG1 kappa, BALB/c), and 36.1.2D (IgM kappa, BALB/c) were obtained by immunization with the T-dependent Ag Isomaltohexaose or isomaltotriose coupled to keyhole limpet hemocyanin or to BSA. Immunochemical characterization of the hybridoma antibodies showed that 16.4.12E and 36.1.2D had cavity-type combining sites, recognizing the terminal non-reducing end of alpha(1----6)dextran, whereas 28.4.10A and 35.8.2H had groove-type sites, recognizing internal linear segments of the dextran. The V region cDNA of the H and L chains of the antibodies were cloned and sequenced. VH of 16.4.12E and VH of 36.1.2D belonged to the X24 and Q52 germ-line gene families, respectively. The VH and V kappa sequences of 16.4.12E and V kappa sequence of 36.1.2D were highly homologous to those of W3129, the only anti-alpha(1----6)dextran mAb with a cavity-type site thus far sequenced; 16.4.12E differed from W3129 in the D, JH, and J kappa. VH genes of 28.4.10A and 35.8.2H were homologous to those of several anti-alpha(1----6)dextrans with groove-type sites, but belonged to the J558 germ-line gene family, differed from the other J558 anti-alpha(1----6)dextrans, probably representing a different germ-line subfamily. The L chain sequence of 28.4.10A encoded by V kappa-Ars and J kappa 2 was almost identical to other groove-type anti-alpha(1----6)dextrans obtained by immunizing with the T-independent glycolipid Ag, stearyl-isomaltotetraose. Use of T-dependent Ag such as isomaltosyl oligosaccharide-protein conjugates provides an additional parameter for probing the fine structure of antibody combining sites and evaluating the V-gene repertoire of anti-alpha(1----6)dextrans.

Specificity of the glucan-binding lectin of Streptococcus cricetus.[Pubmed:3397177]

Infect Immun. 1988 Aug;56(8):1864-72.

The specificity of the glucan-binding lectin (GBL) of Streptococcus cricetus AHT was determined. Examination of the kinetics of aggregation of cell suspensions with glucans containing various percentages of alpha-1,6, alpha-1,4, alpha-1,3, and alpha-1,2 anomeric linkages revealed that only glucans with at least 80% alpha-1,6 linkages promoted strong aggregation. Moreover, only linear glucans with molecular weights greater than 5 X 10(5) were capable of causing rapid aggregation of the bacteria. The lectin was observed to be present on S. cricetus strains, on Streptococcus sobrinus, and on several Streptococcus mutants strains. Preincubation of suspensions of S. cricetus AHT with glucan T10 (molecular weight of 10,000) before the addition of high-molecular-weight glucan resulted in competitive inhibition in a concentration-dependent manner. Inhibition was achieved also with isomaltopentaose, Isomaltohexaose, and isomaltooctaose, but at higher concentrations than glucan T10. In contrast, no inhibition was observed with maltoheptaose, providing additional evidence for the specificity of GBL. Treatment of suspensions of S. cricetus AHT with trypsin before and after aggregation with high-molecular-weight glucan revealed a substantial level of protection of GBL when in a bound state. Collectively, these results indicated that GBL has an absolute affinity for glucans rich in alpha-1,6 linkages and possesses an active site which recognizes internal sequences and accommodates isomaltosaccharides of at least nine residues. This unusual specificity may contribute to the colonization of S. cricetus, S. sobrinus, and S. mutans in glucan-containing plaque in the oral cavity.

Immunochemical studies of conjugates of isomaltosyl oligosaccharides to lipid: production and characterization of mouse hybridoma antibodies specific for stearyl-isomaltosyl oligosaccharides.[Pubmed:2415810]

Mol Immunol. 1985 Sep;22(9):1021-37.

Twelve C57BL/6J hybridoma clones, 9, 2 and 1 from mice immunized with stearyl-isomaltotetraose, stearyl-isomaltopentaose and stearyl-Isomaltohexaose respectively were characterized. Seven produced IgA and 5 IgM. The specificities and sizes of their combining sites were determined by quantitative precipitin and precipitin inhibition assays. All 12 hybridoma antibodies precipitated with alpha 1----6 dextran B512 and linear dextran LD7, indicating that they recognize an internal -Glc alpha 1----6Glc alpha 1----6Glc- determinant. This in contrast with the results with rabbit antisera obtained in response to the same immunogen which recognize the non-reducing terminal determinant Glc alpha 1----6Glc alpha 1----6Glc-. Of the 12 hybridoma antibodies, 1 has an antibody combining site complementary to 4 alpha 1----6-linked glucoses while others have combining sites complementary to Isomaltohexaose or isomaltoheptaose. The large combining-site sizes found in C57BL/6 hybridoma clones may be related to the pre-existing clonal repertoire in this strain. Binding constants of monomers of these antibodies for dextran B512 and isomaltoheptaose determined by affinity electrophoresis range from 1.4 X 10(3) to 4.6 X 10(5) ml/g and from 1.2 X 10(3) to 3.5 X 10(4) M-1 respectively, which is consistent with previous studies in the anti-dextran B512 system. The use of synthetic glycolipids as antigens enables us to study the gene control of antibody responses to glycolipids and to investigate the combining-site specificities of antibodies to a single antigenic determinant. Results so far show that all 12 hybridoma proteins are different despite the simplicity of the antigens. The findings provide further insight into the specificity of antibody combining sites.