Maltooctaose

Thermodynamic Modeling and Experimental Data Reveal That Sugars Stabilize Proteins According to an Excluded Volume Mechanism.[Pubmed:37466340]

J Am Chem Soc. 2023 Aug 2;145(30):16678-16690.

We present a new thermodynamic model to investigate the relative effects of excluded volume and soft interaction contributions in determining whether a cosolute will either destabilize or stabilize a protein in solution. This model is unique in considering an atomistically detailed model of the protein and accounting for the preferential accumulation/exclusion of the osmolyte molecules from the protein surface. Importantly, we use molecular dynamics simulations and experiments to validate the model. The experimental approach presents a unique means of decoupling excluded volume and soft interaction contributions using a linear polymeric series of cosolutes with different numbers of glucose subunits, from 1 (glucose) to 8 (Maltooctaose), as well as an 8-mer of glucose units in the closed form (gamma-CD). By studying the stabilizing effect of cosolutes along this polymeric series using lysozyme as a model protein, we validate the thermodynamic model and show that sugars stabilize proteins according to an excluded volume mechanism.

The Structure of Maltooctaose-Bound Escherichia coli Branching Enzyme Suggests a Mechanism for Donor Chain Specificity.[Pubmed:37298853]

Molecules. 2023 May 27;28(11):4377.

Glycogen is the primary storage polysaccharide in bacteria and animals. It is a glucose polymer linked by alpha-1,4 glucose linkages and branched via alpha-1,6-linkages, with the latter reaction catalyzed by branching enzymes. Both the length and dispensation of these branches are critical in defining the structure, density, and relative bioavailability of the storage polysaccharide. Key to this is the specificity of branching enzymes because they define branch length. Herein, we report the crystal structure of the Maltooctaose-bound branching enzyme from the enterobacteria E. coli. The structure identifies three new malto-oligosaccharide binding sites and confirms oligosaccharide binding in seven others, bringing the total number of oligosaccharide binding sites to twelve. In addition, the structure shows distinctly different binding in previously identified site I, with a substantially longer glucan chain ordered in the binding site. Using the donor oligosaccharide chain-bound Cyanothece branching enzyme structure as a guide, binding site I was identified as the likely binding surface for the extended donor chains that the E. coli branching enzyme is known to transfer. Furthermore, the structure suggests that analogous loops in branching enzymes from a diversity of organisms are responsible for branch chain length specificity. Together, these results suggest a possible mechanism for transfer chain specificity involving some of these surface binding sites.

One-step synthesis of glycogen-type polysaccharides from maltooctaose and its structural characteristics.[Pubmed:35287897]

Carbohydr Polym. 2022 May 15;284:119175.

The one-step synthesis of glycogen-type polysaccharides from Maltooctaose (G8) was accomplished based on the new findings of the catalytic mechanism of glycogen branching enzymes (GBEs) from Vibrio vulnificus, Deinococcus geothermalis, and Escherichia coli. GBEs from D. geothermalis and E. coli used maltononaose and maltotridecaose as the minimum, respectively, while V. vulnificus GBE (VvGBE) catalyzed the surprisingly small substrate, G8, into polysaccharides. Intriguingly, all three GBEs catalyzed alpha-1,4-transglycosylation at the early reaction stage of transglycosylation. VvGBE successfully converted the smallest substrate (G8) into two highly branched polysaccharides (HBP), in which the big polysaccharide (1.49 x 10(5) Da) exhibited structural properties similar to glycogen. Both HBPs had similar side chain distribution with a very short average degree of polymerization compared with mussel glycogen. As a molecular biology reagent, these nucleotide-free HBPs significantly increased the mRNA extraction efficiency of mammalian cells. Our results provide a new approach to the synthesis of novel polysaccharides.

Analysis of oligosaccharides from Panax ginseng by using solid-phase permethylation method combined with ultra-high-performance liquid chromatography-Q-Orbitrap/mass spectrometry.[Pubmed:33192120]

J Ginseng Res. 2020 Nov;44(6):775-783.

BACKGROUND: The reports about valuable oligosaccharides in ginseng are quite limited. There is an urgent need to develop a practical procedure to detect and analyze ginseng oligosaccharides. METHODS: The oligosaccharide extracts from ginseng were permethylated by solid-phase methylation method and then were analyzed by ultra-high-performance liquid chromatography-Q-Orbitrap/MS. The sequence, linkage, and configuration information of oligosaccharides were determined by using accurate m/z value and tandem mass information. Several standard references were used to further confirm the identification. The oligosaccharide composition in white ginseng and red ginseng was compared using a multivariate statistical analysis method. RESULTS: The nonreducing oligosaccharide erlose among 12 oligosaccharides identified was reported for the first time in ginseng. In the comparison of the oligosaccharide extracts from white ginseng and red ginseng, a clear separation was observed in the partial least squares-discriminate analysis score plot, indicating the sugar differences in these two kinds of ginseng samples. The glycans with variable importance in the projection value large than 1.0 were considered to contribute most to the classification. The contents of oligosaccharides in red ginseng were lower than those in white ginseng, and the contents of maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, Maltooctaose, maltononaose, sucrose, and erlose decreased significantly (p < 0.05) in red ginseng. CONCLUSION: A solid-phase methylation method combined with liquid chromatography-tandem mass spectrometry was successfully applied to analyze the oligosaccharides in ginseng extracts, which provides the possibility for holistic evaluation of ginseng oligosaccharides. The comparison of oligosaccharide composition of white ginseng and red ginseng could help understand the differences in pharmacological activities between these two kinds of ginseng samples from the perspective of glycans.

On-Tissue Derivatization with Girard's Reagent P Enhances N-Glycan Signals for Formalin-Fixed Paraffin-Embedded Tissue Sections in MALDI Mass Spectrometry Imaging.[Pubmed:32865977]

Anal Chem. 2020 Oct 6;92(19):13361-13368.

Glycosylation is a major protein post-translational modification whose dysregulation has been associated with many diseases. Herein, an on-tissue chemical derivatization strategy based on positively charged hydrazine reagent (Girard's reagent P) coupled with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed for analysis of N-glycans from FFPE treated tissue sections. The performance of the proposed approach was evaluated by analysis of monosaccharides, oligosaccharides, N-glycans released from glycoproteins, as well as MS imaging of N-glycans from human cancer tissue sections. The results demonstrated that the signal-to-noise ratios for target saccharides were notably improved after chemical derivatization, in which signals were enhanced by 230-fold for glucose and over 28-fold for Maltooctaose. Improved glycome coverage was obtained for N-glycans derived from glycoproteins and tissue samples after chemical derivatization. Furthermore, on-tissue derivatization was applied for MALDI-MSI of N-glycans from human laryngeal cancer and ovarian cancer tissues. Differentially expressed N-glycans among the tumor region, adjacent normal tissue region, and tumor proximal collagen stroma region were imaged, revealing that high-mannose type N-glycans were predominantly expressed in the tumor region. Overall, our results indicate that the on-tissue labeling strategy coupled with MALDI-MSI shows great potential to spatially characterize N-glycan expression within heterogeneous tissue samples with enhanced sensitivity. This study provides a promising approach to better understand the pathogenesis of cancer related aberrant glycosylation, which is beneficial to the design of improved clinical diagnosis and therapeutic strategies.

Efficient Cleavage of Permethylated Cyclodextrins.[Pubmed:31458809]

ACS Omega. 2018 Jun 11;3(6):6279-6282.

The cleavage of a permethylated alpha-cyclodextrin, that is, purity and chemical yield of a maltohexaose derivative thus obtained, was decisively affected by the reaction time. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry spectra with synthetic data enable us to improve the cleavage reaction more efficiently. The optimized conditions developed in the present study allow us to cleave the permethylated gamma-cyclodextrin to afford a Maltooctaose derivative as a new synthetic building block in a high chemical yield.

GH57 amylopullulanase from Desulfurococcus amylolyticus JCM 9188 can make highly branched cyclodextrin via its transglycosylation activity.[Pubmed:29685348]

Enzyme Microb Technol. 2018 Jul;114:15-21.

Desulfurococcus amylolyticus is an anaerobic and hyperthermophilic crenarchaeon that can use various carbohydrates as energy sources. We found a gene encoding a glycoside hydrolase family 57 amylolytic enzymes (DApu) in a putative carbohydrate utilization gene cluster in the genome of D. amylolyticus. This gene has an open reading frame of 1,878 bp and consists of 626 amino acids with a molecular mass of 71 kDa. Recombinant DApu (rDApu) completely hydrolyzed pullulan to maltotriose by attacking alpha-1,6-glycosidic linkages, and was able to produce glucose and maltose from soluble starch and amylopectin. Although rDApu showed no activity toward alpha-cyclodextrin (CD) and beta-CD, Maltooctaose (G8) was detected from reaction with gamma-CD. The highest activity of rDApu was measured at pH 5.0 and 95  degrees C. The half-life of rDApu was 12.7 h at 95  degrees C and 27 min at 98  degrees C. Interestingly, rDApu was able to transfer a maltose unit to 6-O-alpha-maltosyl-beta-CD via transglycosylation. Structure analysis using MALDI-TOF/TOF MS and nuclear magnetic resonance revealed that the new transglycosylated products were 6(1), 6(4)-di-O-maltosyl-beta-CD and 6(1), 6(3), 6(5)-tri-O-maltosyl-beta-CD.

Purification and characterization of a chloride ion-dependent alpha-glucosidase from the midgut gland of Japanese scallop (Patinopecten yessoensis).[Pubmed:26645800]

Biosci Biotechnol Biochem. 2016;80(3):479-85.

Marine glycoside hydrolases hold enormous potential due to their habitat-related characteristics such as salt tolerance, barophilicity, and cold tolerance. We purified an alpha-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 degrees 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.

Introducing transglycosylation activity in Bacillus licheniformis alpha-amylase by replacement of His235 with Glu.[Pubmed:25117441]

Biochem Biophys Res Commun. 2014 Sep 5;451(4):541-7.

To understand the role of His and Glu in the catalytic activity of Bacillus licheniformis alpha-amylase (BLA), His235 was replaced with Glu. The mutant enzyme, H235E, was characterized in terms of its mode of action using labeled and unlabeled Maltooctaose (Glc8). H235E predominantly produced maltotridecaose (Glc13) from Glc8, exhibiting high substrate transglycosylation activity, with Km=0.38mM and kcat/Km=20.58mM(-1)s(-1) for hydrolysis, and Km2=18.38mM and kcat2/Km2=2.57mM(-1)s(-1) for transglycosylation, while the wild-type BLA exhibited high hydrolysis activity exclusively. Glu235-located on a wide open groove near subsite +1-is likely involved in transglycosylation via formation of an alpha-1,4-glycosidic linkage and may recognize and stabilize the non-reducing end glucose of the acceptor molecule.

Experimental evidence for a 9-binding subsite of Bacillus licheniformis thermostable alpha-amylase.[Pubmed:24440349]

FEBS Lett. 2014 Feb 14;588(4):620-4.

The action pattern of Bacillus licheniformis thermostable alpha-amylase (BLA) was analyzed using a series of (14)C-labeled and non-labeled maltooligosaccharides from maltose (G2) to maltododecaose (G12). Maltononaose (G9) was the preferred substrate, and yielded the smallest Km=0.36 mM, the highest kcat=12.86 s(-1), and a kcat/Km value of 35.72 s(-1) mM(-1), producing maltotriose (G3) and maltohexaose (G6) as the major product pair. Maltooctaose (G8) was hydrolyzed into two pairs of products: G3 and maltopentaose (G5), and G2 and G6 with cleavage frequencies of 0.45 and 0.30, respectively. Therefore, we propose a model with nine subsites: six in the terminal non-reducing end-binding site and three at the reducing end-binding site in the binding region of BLA.

Enzymatic synthesis of Acarviosyl-maltooligosaccharides using disproportionating enzyme 1.[Pubmed:23391922]

Biosci Biotechnol Biochem. 2013;77(2):312-9.

Acarbose is a pseudo-tetrasaccharide and one of the most effective inhibitors of glycoside hydrolases. Its derivatives, acarviosyl-maltooligosaccharides, which have longer maltooligosaccharide parts than the maltose unit of acarbose, were synthesized using a disproportionating enzyme partially purified from adzuki cotyledons. The enzyme was identified as a typical type-1 disproportionating enzyme (DPE1) by primary structure analysis. It produced six compounds from 100 mM acarbose and 7.5% (w/v) of maltotetraose-rich syrup. The masses of the six products were confirmed to accord with acarviosyl-maltooligosaccharides with the degrees of polymerization = 5-10 (AC5-AC10) by electrospray ionization mass spectrometry. (1)H and (13)C NMR spectra indicated that AC5-AC10 were alpha-acarviosyl-(1-->4)-maltooligosaccharide, which have maltotriose-Maltooctaose respectively in the maltooligosaccharide part. A predominance of AC7 in the products at the early stage of the reaction indicated that DPE1 catalyzes the transfer of the acarviosyl-glucose moiety from acarbose to the acceptors. ACn can be useful tools as new inhibitors of glycoside hydrolases.

An extremely thermostable amylopullulanase from Staphylothermus marinus displays both pullulan- and cyclodextrin-degrading activities.[Pubmed:23001056]

Appl Microbiol Biotechnol. 2013 Jun;97(12):5359-69.

A gene encoding an amylopullulanase of the glycosyl hydrolase (GH) family 57 from Staphylothermus marinus (SMApu) was heterologously expressed in Escherichia coli. SMApu consisted of 639 amino acids with a molecular mass of 75.3 kDa. It only showed maximal amino acid identity of 17.1 % with that of Pyrococcus furiosus amylopullulanase in all identified amylases. Not like previously reported amylopullulanases, SMApu has no signal peptide but contains a continuous GH57N_Apu domain. It had the highest catalytic efficiency toward pullulan (k cat/K m , 342.34 s(-1) mL mg(-1)) and was extremely thermostable with maximal pullulan-degrading activity (42.1 U/mg) at 105 degrees C and pH 5.0 and a half-life of 50 min at 100 degrees C. Its activity increased to 116 % in the presence of 5 mM CaCl2. SMApu could also degrade cyclodextrins, which are resistant to the other amylopullulanases. The initial hydrolytic products from pullulan, gamma-CD, and 6-O-maltooligosyl-beta-CD were [6)-alpha-D-Glcp-(1 --> 4)-alpha-D-Glcp-(1 --> 4)-alpha-D-Glcp-(1-->]n, Maltooctaose, and single maltooligosaccharide plus beta-CD, respectively. The final hydrolytic products from above-mentioned substrates were maltose and glucose. These results confirm that SMApu is a novel amylopullulanase of the family GH57 possessing the cyclodextrin-degrading activity of cyclomaltodextrinase.

Structural characterization and immunological activities of the water-soluble oligosaccharides isolated from the Panax ginseng roots.[Pubmed:22183124]

Planta. 2012 Jun;235(6):1289-97.

Water-soluble ginseng oligosaccharides (designated as WGOS) with a degree of polymerization ranging from 2 to 10 were obtained from warm-water extract of Panax ginseng roots, and fractionated into five purified fractions (i.e., WGOS-0, WGOS-1, WGOS-2, WGOS-3, and WGOS-4) by gel-filtration chromatography. In order to ascertain the monosaccharide residues in the WGOS, a technique that combines acid hydrolysis and high-performance liquid chromatography was employed. It was found that only glucose residues were present in the WGOS. Fourier transform infrared spectroscopy and electrospray ionization tandem mass spectrometry provided the sequence, linkage, and configuration information. It is noteworthy that alpha-Glcp-(1 --> 6)-alpha-Glcp, alpha-Glcp-(1 --> 6)-alpha-Glcp-(1 --> 4)-alpha-Glcp, alpha-Glcp-(1 --> 6)-alpha-Glcp-(1 --> 6)-alpha-Glcp-(1 --> 4)-alpha-Glcp, and other six malto-oligosaccharides (i.e., maltopentaose, maltohexaose, maltoheptaose, Maltooctaose, maltononaose, and maltodecaose) were detected in ginseng. Preliminary immunological tests in vitro indicated that WGOS were potent B and T-cell stimulators and WGOS-1 has the highest immunostimulating effect on lymphocyte proliferation among those purified fractions. It is hoped that the WGOS will be developed into functional food or medicine.

Multiple glycogen-binding sites in eukaryotic glycogen synthase are required for high catalytic efficiency toward glycogen.[Pubmed:21835915]

J Biol Chem. 2011 Sep 30;286(39):33999-4006.

Glycogen synthase is a rate-limiting enzyme in the biosynthesis of glycogen and has an essential role in glucose homeostasis. The three-dimensional structures of yeast glycogen synthase (Gsy2p) complexed with Maltooctaose identified four conserved maltodextrin-binding sites distributed across the surface of the enzyme. Site-1 is positioned on the N-terminal domain, site-2 and site-3 are present on the C-terminal domain, and site-4 is located in an interdomain cleft adjacent to the active site. Mutation of these surface sites decreased glycogen binding and catalytic efficiency toward glycogen. Mutations within site-1 and site-2 reduced the V(max)/S(0.5) for glycogen by 40- and 70-fold, respectively. Combined mutation of site-1 and site-2 decreased the V(max)/S(0.5) for glycogen by >3000-fold. Consistent with the in vitro data, glycogen accumulation in glycogen synthase-deficient yeast cells (Deltagsy1-gsy2) transformed with the site-1, site-2, combined site-1/site-2, or site-4 mutant form of Gsy2p was decreased by up to 40-fold. In contrast to the glycogen results, the ability to utilize Maltooctaose as an in vitro substrate was unaffected in the site-2 mutant, moderately affected in the site-1 mutant, and almost completely abolished in the site-4 mutant. These data show that the ability to utilize Maltooctaose as a substrate can be independent of the ability to utilize glycogen. Our data support the hypothesis that site-1 and site-2 provide a "toehold mechanism," keeping glycogen synthase tightly associated with the glycogen particle, whereas site-4 is more closely associated with positioning of the nonreducing end during catalysis.