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International Journal of Molecular... Jul 2015Three kinds of polysaccharides, namely, BSP1A, BSP2A, and BSP3B, were isolated from raw bamboo shoot (Dendrocalamus latiflorus) after purification and classification by...
Three kinds of polysaccharides, namely, BSP1A, BSP2A, and BSP3B, were isolated from raw bamboo shoot (Dendrocalamus latiflorus) after purification and classification by DEAE cellulose-52 (ion-exchange chromatography) and Sephadex G-50. The molecular weights of BSP1A, BSP2A, and BSP3B were 10.2, 17.0 and 20.0 kDa, respectively, which were measured through GPC (gel performance chromatography) methods. BSP1A contained arabinose, glucose, and galactose in a molar ratio of 1.0:40.6:8.7. BSP2A and BSP3B contained arabinose, xylose, glucose, and galactose in molar ratios of 6.6:1.0:5.2:10.4 and 8.5:1.0:5.1:11.1, respectively. The existence of the O-glycopeptide bond in BSP1A, BSP2A, and BSP3B was demonstrated by β-elimination reaction. FTIR spectra of the three polysaccharides showed that both BSP2A and BSP3B contained β-D-pyranose sugar rings. However, BSP1A exhibited both β-D-pyranose and α-D-pyranose sugar rings. Congo red test indicated that BSP1A and BSP2A displayed triple helix structures, but BSP3B did not. NMR spectroscopy revealed that BSP1A may exhibit a β-1,6-Glucan pyran type as the main link, and few 1,6-glycosidic galactose pyranose and arabinose bonds were connected; BSP2A mainly demonstrated → 5)β-Ara(1 → and → 3)β-Gal(1 → connection. Furthermore, BSP3B mainly presented → 3)β-Glu(1 → and → 3)β-Gal(1 → connection and may also contain few other glycosidic bonds.
Topics: Arabinose; Carbohydrate Conformation; Chromatography, Gas; Chromatography, Gel; Chromatography, Ion Exchange; Galactose; Glucose; Molecular Weight; Plant Shoots; Poaceae; Polysaccharides; Spectroscopy, Fourier Transform Infrared
PubMed: 26184163
DOI: 10.3390/ijms160715560 -
Fungal Genetics and Biology : FG & B Feb 2019l-Arabinose and d-galactose are the principal constituents of l-arabinogalactan, and also co-occur in other hemicelluloses and pectins. In this work we hypothesized that...
l-Arabinose and d-galactose are the principal constituents of l-arabinogalactan, and also co-occur in other hemicelluloses and pectins. In this work we hypothesized that similar to the induction of relevant glycoside hydrolases by monomers liberated from these plant heteropolymers, their respective catabolisms in saprophytic and phytopathogenic fungi may respond to the presence of the other sugar to promote synergistic use of the complex growth substrate. We showed that these two sugars are indeed consumed simultaneously by Aspergillus nidulans, while l-arabinose is utilised faster in the presence than in the absence of d-galactose. Furthermore, the first two genes of the Leloir pathway for d-galactose catabolism - encoding d-galactose 1-epimerase and galactokinase - are induced more rapidly by l-arabinose than by d-galactose eventhough deletion mutants thereof grow as well as a wild type strain on the pentose. d-Galactose 1-epimerase is hyperinduced by l-arabinose, d-xylose and l-arabitol but not by xylitol. The results suggest that in A. nidulans, l-arabinose and d-xylose - both requiring NADPH for their catabolisation - actively promote the enzyme infrastructure necessary to convert β-d-galactopyranose via the Leloir pathway with its α-anomer specific enzymes, into β-d-glucose-6-phosphate (the starting substrate of the oxidative part of the pentose phosphate pathway) even in the absence of d-galactose.
Topics: Arabinose; Aspergillus nidulans; Galactans; Galactose; Gene Expression Regulation, Fungal; Metabolic Networks and Pathways; Metabolism; Pectins; Polysaccharides; UDPglucose 4-Epimerase; Xylose
PubMed: 30496805
DOI: 10.1016/j.fgb.2018.11.004 -
Applied and Environmental Microbiology Feb 2018Pentoses, including xylose and arabinose, are the second most prevalent sugars in lignocellulosic biomass that can be harnessed for biological conversion. Although has...
Pentoses, including xylose and arabinose, are the second most prevalent sugars in lignocellulosic biomass that can be harnessed for biological conversion. Although has emerged as a promising industrial microorganism for production of high-value chemicals and biofuels, its native pentose metabolism is poorly understood. Our previous study demonstrated that (ATCC MYA-2613) has endogenous enzymes for d-xylose assimilation, but inefficient xylitol dehydrogenase causes to assimilate xylose poorly. In this study, we investigated the functional roles of native sugar-specific transporters for activating the dormant pentose metabolism in By screening a comprehensive set of 16 putative pentose-specific transporters, we identified two candidates, YALI0C04730p and YALI0B00396p, that enhanced xylose assimilation. The engineered mutants YlSR207 and YlSR223, overexpressing YALI0C04730p and YALI0B00396p, respectively, improved xylose assimilation approximately 23% and 50% in comparison to YlSR102, a parental engineered strain overexpressing solely the native xylitol dehydrogenase gene. Further, we activated and elucidated a widely unknown native l-arabinose assimilation pathway in through transcriptomic and metabolic analyses. We discovered that can coconsume xylose and arabinose, where arabinose utilization shares transporters and metabolic enzymes of some intermediate steps of the xylose assimilation pathway. Arabinose assimilation is synergistically enhanced in the presence of xylose, while xylose assimilation is competitively inhibited by arabinose. l-Arabitol dehydrogenase is the rate-limiting step responsible for poor arabinose utilization in Overall, this study sheds light on the cryptic pentose metabolism of and, further, helps guide strain engineering of for enhanced assimilation of pentose sugars. The oleaginous yeast is a promising industrial-platform microorganism for production of high-value chemicals and fuels. For decades since its isolation, has been known to be incapable of assimilating pentose sugars, xylose and arabinose, that are dominantly present in lignocellulosic biomass. Through bioinformatic, transcriptomic, and enzymatic studies, we have uncovered the dormant pentose metabolism of Remarkably, unlike most yeast strains, which share the same transporters for importing hexose and pentose sugars, we discovered that possesses the native pentose-specific transporters. By overexpressing these transporters together with the rate-limiting d-xylitol and l-arabitol dehydrogenases, we activated the dormant pentose metabolism of Overall, this study provides a fundamental understanding of the dormant pentose metabolism of and guides future metabolic engineering of for enhanced conversion of pentose sugars to high-value chemicals and fuels.
Topics: Arabinose; Biofuels; Biomass; Computational Biology; Ethanol; Fermentation; Glucose; Metabolic Engineering; Metabolic Networks and Pathways; Pentoses; Sugar Alcohols; Xylose; Yarrowia
PubMed: 29150499
DOI: 10.1128/AEM.02146-17 -
Biochemical and Biophysical Research... Jul 2022The mitogen-activated protein kinase (MAPK), as a major member in MAPK cascade, has been shown to play an important role in plant development and growth. However, little...
The mitogen-activated protein kinase (MAPK), as a major member in MAPK cascade, has been shown to play an important role in plant development and growth. However, little is known about the function of MAPK in regulating cell wall synthesis/metabolism. In this study, we found that the l-arabinose content in mpk4 mutant was much higher compared to those in wild type, mpk3 and mpk6 mutants, whereas overexpressing MPK4 in Arabidopsis obviously decreased the l-arabinose content of cell wall. Furthermore, loss of function in MPK4 significantly decreased the expression of l-arabinose synthesis/metabolism-related gene MUR10, but did not affect the expressions of the other genes (MUR4, MUR5, UXT1 and ARAD1). Moreover, knock-out of MPK4 significantly decreased the cellulose content. These results suggest that MPK4 negatively regulates the l-arabinose synthesis of cell wall by likely modulating the expression of MUR10.
Topics: Arabidopsis; Arabidopsis Proteins; Arabinose; Cell Wall; Gene Expression Regulation, Plant; Mitogen-Activated Protein Kinases
PubMed: 35526489
DOI: 10.1016/j.bbrc.2022.04.116 -
Glycobiology Jul 2019The endoplasmic reticulum (ER) contains both α-glucosidases and α-mannosidases which process the N-linked oligosaccharides of newly synthesized glycoproteins and...
The endoplasmic reticulum (ER) contains both α-glucosidases and α-mannosidases which process the N-linked oligosaccharides of newly synthesized glycoproteins and thereby facilitate polypeptide folding and glycoprotein quality control. By acting as structural mimetics, iminosugars can selectively inhibit these ER localized α-glycosidases, preventing N-glycan trimming and providing a molecular basis for their therapeutic applications. In this study, we investigate the effects of a panel of nine iminosugars on the actions of ER luminal α-glucosidase I and α-glucosidase II. Using ER microsomes to recapitulate authentic protein N-glycosylation and oligosaccharide processing, we identify five iminosugars that selectively inhibit N-glycan trimming. Comparison of their inhibitory activities in ER microsomes against their effects on purified ER α-glucosidase II, suggests that 3,7a-diepi-alexine acts as a selective inhibitor of ER α-glucosidase I. The other active iminosugars all inhibit α-glucosidase II and, having identified 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) as the most effective of these compounds, we use in silico modeling to understand the molecular basis for this enhanced activity. Taken together, our work identifies the C-3 substituted pyrrolizidines casuarine and 3,7a-diepi-alexine as promising "second-generation" iminosugar inhibitors.
Topics: Animals; Arabinose; Dogs; Endoplasmic Reticulum; Glycoside Hydrolase Inhibitors; Humans; Imino Furanoses; Mice; Microsomes; Pyrrolizidine Alkaloids; Sugar Alcohols; alpha-Glucosidases
PubMed: 30976784
DOI: 10.1093/glycob/cwz029 -
International Journal of Biological... May 2018Two components ginger polysaccharide 1 (GP1) and ginger polysaccharide 2 (GP2) were extracted. The results showed that the molecular weights of GP1 and GP2 were...
Two components ginger polysaccharide 1 (GP1) and ginger polysaccharide 2 (GP2) were extracted. The results showed that the molecular weights of GP1 and GP2 were 6128 Da and 12,619 Da, respectively. The composition and proportion of GP1 and GP2 were mannose, glucose and galactose in a molar ratio of 4.96: 92.24: 2.80 and arabinose, mannose, glucose and galactose in a molar ratio of 4.78: 16.70: 61.77: 16.75, respectively, illustrating that GP1 and GP2 were not a kind of homopolysaccharide. GP1 has a three-helix structure, and the structure is closely linked. GP2 contains sulfuric acid groups, and has a high oxidation resistance, its structure is more evacuated and messy.
Topics: Antioxidants; Arabinose; Dietary Carbohydrates; Galactose; Zingiber officinale; Glucose; Mannose; Molecular Structure; Oxidation-Reduction; Polysaccharides; Protein Conformation, alpha-Helical; Sulfuric Acids
PubMed: 29360545
DOI: 10.1016/j.ijbiomac.2018.01.087 -
Plant Physiology Mar 2018The controlled distribution of sugars between assimilate-exporting source tissues and sugar-consuming sink tissues is a key element for plant growth and development....
The controlled distribution of sugars between assimilate-exporting source tissues and sugar-consuming sink tissues is a key element for plant growth and development. Monosaccharide transporters of the SUGAR TRANSPORT PROTEIN (STP) family contribute to the uptake of sugars into sink cells. Here, we report on the characterization of STP7, STP8, and STP12, three previously uncharacterized members of this family in Arabidopsis (). Heterologous expression in yeast () revealed that STP8 and STP12 catalyze the high-affinity proton-dependent uptake of glucose and also accept galactose and mannose. STP12 additionally transports xylose. and are highly expressed in reproductive organs, where their protein products might contribute to sugar uptake into the pollen tube and the embryo sac. and T-DNA insertion lines developed normally, which may point toward functional redundancy with other STPs. In contrast to all other STPs, STP7 does not transport hexoses but is specific for the pentoses l-arabinose and d-xylose. promoter-reporter gene plants showed an expression of especially in tissues with high cell wall turnover, indicating that STP7 might contribute to the uptake and recycling of cell wall sugars. Uptake analyses with radioactive l-arabinose revealed that 11 other STPs are able to transport l-arabinose with high affinity. Hence, functional redundancy might explain the missing-mutant phenotype of two T-DNA insertion lines. Together, these data complete the characterization of the STP family and present the STPs as new l-arabinose transporters for potential biotechnological applications.
Topics: Arabidopsis; Arabidopsis Proteins; Arabinose; DNA, Bacterial; Gene Expression Regulation, Plant; Genes, Reporter; Green Fluorescent Proteins; Monosaccharide Transport Proteins; Plants, Genetically Modified; Saccharomyces cerevisiae; Xylose
PubMed: 29311272
DOI: 10.1104/pp.17.01493 -
MAbs 2017Protein glycosylation is arguably the paramount post-translational modification on recombinant glycoproteins, and highly cited in the literature for affecting the...
Protein glycosylation is arguably the paramount post-translational modification on recombinant glycoproteins, and highly cited in the literature for affecting the physiochemical properties and the efficacy of recombinant glycoprotein therapeutics. Glycosylation of human immunoglobulins follows a reasonably well-understood metabolic pathway, which gives rise to a diverse range of asparagine-linked (N-linked), or serine/threonine-linked (O-linked) glycans. In N-linked glycans, fucose levels have been shown to have an inverse relationship with the degree of antibody-dependent cell-mediated cytotoxicity, and high mannose levels have been implicated in potentially increasing immunogenicity and contributing to less favorable pharmacokinetic profiles. Here, we demonstrate a novel approach to potentially reduce the presence of high-mannose species in recombinant human immunoglobulin preparations, as well as facilitate an approximate 100% replacement of fucosylation with arabinosylation in Chinese hamster ovary cell culture through media supplementation with D-arabinose, an uncommonly used mammalian cell culture sugar substrate. The replacement of fucose with arabinose was very effective and practical to implement, since no cell line engineering or cellular adaptation strategies were required. Arabinosylated recombinant IgGs and the accompanying reduction in high mannose glycans, facilitated a reduction in dendritic cell uptake, increased FcγRIIIa signaling, and significantly increased the levels of ADCC. These aforementioned effects were without any adverse changes to various structural or functional attributes of multiple recombinant human antibodies and a bispecific DVD-Ig. Protein arabinosylation represents an expansion of the N-glycan code in mammalian expressed glycoproteins.
Topics: Animals; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Arabinose; CHO Cells; Cricetulus; Glycosylation; Humans; Immunoglobulin G; Recombinant Proteins
PubMed: 28375048
DOI: 10.1080/19420862.2017.1294295 -
Acta Crystallographica. Section D,... Nov 2014L-Arabinose sugar residues are relatively abundant in plants and are found mainly in arabinan polysaccharides and in other arabinose-containing polysaccharides such as...
L-Arabinose sugar residues are relatively abundant in plants and are found mainly in arabinan polysaccharides and in other arabinose-containing polysaccharides such as arabinoxylans and pectic arabinogalactans. The majority of the arabinose units in plants are present in the furanose form and only a small fraction of them are present in the pyranose form. The L-arabinan-utilization system in Geobacillus stearothermophilus T6, a Gram-positive thermophilic soil bacterium, has recently been characterized, and one of the key enzymes was found to be an intracellular β-L-arabinopyranosidase (Abp). Abp, a GH27 enzyme, was shown to remove β-L-arabinopyranose residues from synthetic substrates and from the native substrates sugar beet arabinan and larch arabinogalactan. The Abp monomer is made up of 448 amino acids, and based on sequence homology it was suggested that Asp197 is the catalytic nucleophile and Asp255 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Abp (at 2.28 Å resolution) and its catalytic mutant Abp-D197A with (at 2.20 Å resolution) and without (at 2.30 Å resolution) a bound L-arabinose product are reported as determined by X-ray crystallography. These structures demonstrate that the three-dimensional structure of the Abp monomer correlates with the general fold observed for GH27 proteins, consisting of two main domains: an N-terminal TIM-barrel domain and a C-terminal all-β domain. The two catalytic residues are located in the TIM-barrel domain, such that their carboxylic functional groups are about 5.9 Å from each other, consistent with a retaining mechanism. An isoleucine residue (Ile67) located at a key position in the active site is shown to play a critical role in the substrate specificity of Abp, providing a structural basis for the high preference of the enzyme towards arabinopyranoside over galactopyranoside substrates. The crystal structure demonstrates that Abp is a tetramer made up of two `open-pincers' dimers, which clamp around each other to form a central cavity. The four active sites of the Abp tetramer are situated on the inner surface of this cavity, all opening into the central space of the cavity. The biological relevance of this tetrameric structure is supported by independent results obtained from size-exclusion chromatography (SEC), dynamic light-scattering (DLS) and small-angle X-ray scattering (SAXS) experiments. These data and their comparison to the structural data of related GH27 enzymes are used for a more general discussion concerning structure-selectivity aspects in this glycoside hydrolase (GH) family.
Topics: Arabinose; Catalytic Domain; Crystallography, X-Ray; Geobacillus stearothermophilus; Glycoside Hydrolases; Models, Molecular; Point Mutation; Protein Conformation; Protein Multimerization; Scattering, Small Angle; Substrate Specificity; X-Ray Diffraction
PubMed: 25372689
DOI: 10.1107/S139900471401863X -
Microbial Cell Factories Aug 2023R. toruloides is an oleaginous yeast, with diverse metabolic capacities and high tolerance for inhibitory compounds abundant in plant biomass hydrolysates. While R....
R. toruloides is an oleaginous yeast, with diverse metabolic capacities and high tolerance for inhibitory compounds abundant in plant biomass hydrolysates. While R. toruloides grows on several pentose sugars and alcohols, further engineering of the native pathway is required for efficient conversion of biomass-derived sugars to higher value bioproducts. A previous high-throughput study inferred that R. toruloides possesses a non-canonical L-arabinose and D-xylose metabolism proceeding through D-arabitol and D-ribulose. In this study, we present a combination of genetic and metabolite data that refine and extend that model. Chiral separations definitively illustrate that D-arabitol is the enantiomer that accumulates under pentose metabolism. Deletion of putative D-arabitol-2-dehydrogenase (RTO4_9990) results in > 75% conversion of D-xylose to D-arabitol, and is growth-complemented on pentoses by heterologous xylulose kinase expression. Deletion of putative D-ribulose kinase (RTO4_14368) arrests all growth on any pentose tested. Analysis of several pentose dehydrogenase mutants elucidates a complex pathway with multiple enzymes mediating multiple different reactions in differing combinations, from which we also inferred a putative L-ribulose utilization pathway. Our results suggest that we have identified enzymes responsible for the majority of pathway flux, with additional unknown enzymes providing accessory activity at multiple steps. Further biochemical characterization of the enzymes described here will enable a more complete and quantitative understanding of R. toruloides pentose metabolism. These findings add to a growing understanding of the diversity and complexity of microbial pentose metabolism.
Topics: Xylose; Arabinose; Pentoses
PubMed: 37537595
DOI: 10.1186/s12934-023-02126-x