| 1. |
- Caspersen, Mikael B., et al.
(författare)
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Phosphorylation sites of Arabidopsis MAP kinase substrate 1 (MKS 1)
- 2007
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639. ; 1774:9, s. 1156-1163
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Tidskriftsartikel (refereegranskat)abstract
- The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified by electrophoresis and gel-staining with ProQ Diamond and the protein was digested by either trypsin or chymotrypsin for maximum sequence coverage to facilitate identification of phosphorylated positions. Prior to analysis by mass spectrometry, samples were either desalted, passed over TiO2 or both for improved phosphopeptide detection. As MAP kinases generally phosphorylate serine or threonine followed by proline (Ser/Thr-Pro), theoretical masses of potentially phosphorylated peptides were calculated and mass spectrometric peaks matching these masses were fragmented and searched for a neutral-loss signal at similar to 98 Da indicative of phosphorylation. Additionally, mass spectrometric peaks present in the MPK4-treated MKS1, but not in the control peptide map of untreated MKS1, were fragmented. Fragmentation spectra were subjected to a MASCOT database search which identified three of the twelve Ser-Pro serine residues (Ser72, Set108, Ser120) in the phosphorylated form.
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| 2. |
- Ejby, Morten, et al.
(författare)
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An atp binding cassette transporter mediates the uptake of α-(1,6)-linked dietary oligosaccharides in bifidobacterium and correlates with competitive growth on these substrates
- 2016
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 291:38, s. 20220-20231
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Tidskriftsartikel (refereegranskat)abstract
- The molecular details and impact of oligosaccharide uptake by distinct human gut microbiota (HGM) are currently not well understood. Non-digestible dietary galacto- and gluco-α-(1,6)-oligosaccharides from legumes and starch, respectively, are preferentially fermented by mainly bifidobacteria and lactobacilli in the human gut. Here we show that the solute binding protein (BlG16BP) associated with an ATP binding cassette (ABC) transporter from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 binds α-(1,6)-linked glucosides and galactosides of varying size, linkage, and monosaccharide composition with preference for the trisaccharides raffinose and panose. This preference is also reflected in the α-(1,6)-galactoside uptake profile of the bacterium. Structures of BlG16BP in complex with raffinose and panose revealed the basis for the remarkable ligand binding plasticity of BlG16BP, which recognizes the nonreducing α-(1,6)-diglycoside in its ligands. BlG16BP homologues occur predominantly in bifidobacteria and a few Firmicutes but lack in other HGMs. Among seven bifidobacterial taxa, only those possessing this transporter displayed growth on α-(1,6)-glycosides. Competition assays revealed that the dominant HGM commensal Bacteroides ovatus was out-competed by B. animalis subsp. lactis Bl-04 in mixed cultures growing on raffinose, the preferred ligand for the BlG16BP. By comparison, B. ovatus mono-cultures grew very efficiently on this trisaccharide These findings suggest that the ABC-mediated uptake of raffinose provides an important competitive advantage, particularly against dominant Bacteroides that lack glycan-specific ABC-transporters. This novel insight highlights the role of glycan transport in defining the metabolic specialization of gut bacteria.
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| 3. |
- Ejby, Morten, et al.
(författare)
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Structural basis for arabinoxylo-oligosaccharide capture by the probiotic Bifidobacterium animalis subsp lactis Bl-04
- 2013
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 90:5, s. 1100-1112
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Tidskriftsartikel (refereegranskat)abstract
- Glycan utilization plays a key role in modulating the composition of the gut microbiota, but molecular insight into oligosaccharide uptake by this microbial community is lacking. Arabinoxylo-oligosaccharides (AXOS) are abundant in the diet, and are selectively fermented by probiotic bifidobacteria in the colon. Here we show how selectivity for AXOS uptake is established by the probiotic strain Bifidobacterium animalis subsp. lactisBl-04. The binding protein BlAXBP, which is associated with an ATP-binding cassette (ABC) transporter that mediates the uptake of AXOS, displays an exceptionally broad specificity for arabinosyl-decorated and undecorated xylo-oligosaccharides, with preference for tri- and tetra-saccharides. Crystal structures of BlAXBP in complex with four different ligands revealed the basis for this versatility. Uniquely, the protein was able to recognize oligosaccharides in two opposite orientations, which facilitates the optimization of interactions with the various ligands. Broad substrate specificity was further enhanced by a spacious binding pocket accommodating decorations at different mainchain positions and conformational flexibility of a lid-like loop. Phylogenetic and genetic analyses show that BlAXBP is highly conserved within Bifidobacterium, but is lacking in other gut microbiota members. These data indicate niche adaptation within Bifidobacterium and highlight the metabolic syntrophy (cross-feeding) among the gut microbiota.
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| 4. |
- Ejby, Morten, et al.
(författare)
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Structural basis for arabinoxylo-oligosaccharide capture by the probiotic Bifidobacterium animalis subsp lactis Bl-04
- 2012
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Ingår i: Journal of Psychopharmacology. - 1461-7285. ; 26:11, s. 1100-1112
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Tidskriftsartikel (refereegranskat)abstract
- Glycan utilization plays a key role in modulating the composition of the gut microbiota, but molecular insight into oligosaccharide uptake by this microbial community is lacking. Arabinoxylo-oligosaccharides (AXOS) are abundant in the diet, and are selectively fermented by probiotic bifidobacteria in the colon. Here we show how selectivity for AXOS uptake is established by the probiotic strain Bifidobacterium animalis subsp. lactisBl-04. The binding protein BlAXBP, which is associated with an ATP-binding cassette (ABC) transporter that mediates the uptake of AXOS, displays an exceptionally broad specificity for arabinosyl-decorated and undecorated xylo-oligosaccharides, with preference for tri- and tetra-saccharides. Crystal structures of BlAXBP in complex with four different ligands revealed the basis for this versatility. Uniquely, the protein was able to recognize oligosaccharides in two opposite orientations, which facilitates the optimization of interactions with the various ligands. Broad substrate specificity was further enhanced by a spacious binding pocket accommodating decorations at different mainchain positions and conformational flexibility of a lid-like loop. Phylogenetic and genetic analyses show that BlAXBP is highly conserved within Bifidobacterium, but is lacking in other gut microbiota members. These data indicate niche adaptation within Bifidobacterium and highlight the metabolic syntrophy (cross-feeding) among the gut microbiota.
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| 5. |
- Granlund, Irene, 1961-
(författare)
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Proteomic analysis of Arabidopsis thaliana
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A complete proteome analysis of the chloroplast stroma, using 2D-PAGE, from spinach and Arabidopsis was performed. To improve the identification of proteins a computer program named SPECLUST was used. In SPECLUST, peak masses that are similar in many spots cluster together because they originate from the same protein with different locations on the gel. Within this program peaks in a cluster can be investigated in detail by peaks-in-common, and the unidentified masses that differ between spots in a cluster could be caused by protein modifications, which was analysed further by MS/MS. The thylakoid is an internal membrane system in the chloroplast where protein complexes involved in photosynthesis are housed. Enclosed in the thylakoid membrane is the chloroplast lumen, with a proteome estimated to contain 80-200 different proteins. Because the chloroplast lumen is close to the photosynthesis machinery in the plant, one can expect that the lumen proteome will change depending on if the plant is dark or light adapted. DIGE analysis of lumen proteins found that 15 lumen proteins show increased relative abundance in light-adapted plants. In addition co-expression analysis of lumen protein genes suggests that the lumen protein genes are uniformly transcriptionally regulated, not only by light but in a general manner. Plastocyanin is one of the proteins involved in the electron transfer in photosynthesis. Two homologous plastocyanin isoforms are encoded by the genes PETE1 and PETE2 in the nuclear genome of Arabidopsis, where PETE2 is the more abundant isoform. Knockout mutants of each of the plastocyanin isoforms shows that a 90% reduction of plastocyanin levels affects rates of photosynthesis and growth only slightly. A corresponding over-expression of plastocyanin in each of the two knockout mutants results in essentially wild-type photosynthetic performance. Reduced plastocyanin levels make the plant sensitive to Cu stress and therefore plastocyanin plays a major role as a Cu sink. A by-product of photosynthesis is hydrogen peroxide, which may be harmful for the plant. The discovery that an abundant protein found in the chloroplast lumen, TL29, shared sequence homology to Ascorbate Peroxidase (APX) was therefore of interest. We have evidence that TL29 is not an APX protein; it lacks the heme-binding active site and shows no activity. TL29 is located in the grana region and is electrostaticaly attached to the thylakoid membrane. It has four isoforms, with different pIs, both in the native and denatured form. It has no interaction with ascorbate, when compared to raAPX1. TL29 has two cysteine residues and one of them seems to have redox-regulated function, proposing that it may interact with other proteins close to PSII.
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| 6. |
- Larsbrink, Johan, 1982-
(författare)
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Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The focus of this thesis is a comparative study of approaches in discovery of carbohydrate-active enzymes (CAZymes). CAZymes synthesise, bind to, and degrade all the multitude of carbohydrates found in nature. As such, when aiming for sustainable methods to degrade plant biomass for the generation of biofuels, for which there is a strong drive in society, CAZymes are a natural source of environmentally friendly molecular tools.In nature, microorganisms are the principal degraders of carbohydrates. Not only do they degrade plant matter in forests and aquatic habitats, but also break down the majority of carbohydrates ingested by animals. These symbiotic microorganisms, known as the microbiota, reside in animal digestive tracts in immense quantities, where one of the key nutrient sources is complex carbohydrates. Thus, microorganisms are a plentiful source of CAZymes, and strategies in the discovery of new enzymes from bacterial sources have been the basis for the work presented here, combined with biochemical characterisation of several enzymes.Novel enzymatic activities for the glycoside hydrolase family 31 have been described as a result of the initial projects of the thesis. These later evolved into projects studying bacterial multi-gene systems for the partial or complete degradation of the heterogeneous plant polysaccharide xyloglucan. These systems contain, in addition to various hydrolytic CAZymes, necessary binding-, transport-, and regulatory proteins. The results presented here show, in detail, how very complex carbohydrates can efficiently be degraded by bacterial enzymes of industrial relevance.
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| 7. |
- Madsen, Jan Busk, et al.
(författare)
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Structural and Mechanical Properties of Thin Films of Bovine Submaxillary Mucin (BSM) vs. Porcine Gastric Mucin (PGM) on a Hydrophobic Surface in Aqueous Solutions
- 2016
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 32:38, s. 9687-9696
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Tidskriftsartikel (refereegranskat)abstract
- The structural and mechanical properties of thin films generated from two types of mucins, namely, bovine submaxillary mucin (BSM) and porcine gastric mucin (PGM) in aqueous environment were investigated with several bulk and surface analytical techniques. Both mucins generated hydrated films on hydrophobic polydimethylsiloxane (PDMS) surfaces from spontaneous adsorption arising from their amphiphilic characteristic. However, BSM formed more elastic films than PGM at neutral pH condition. This structural difference was manifested from the initial film formation processes to the responses to shear stresses applied to the films. Acidification of environmental pH led to strengthening the elastic character of BSM films with increased adsorbed mass, whereas an opposite trend was observed for PGM films. We propose that this contrast originates from that negatively charged motifs are present for both the central and terminal regions of BSM molecule, whereas a similar magnitude of negative charges is localized at the termini of PGM molecule. Given that hydrophobic motifs acting as an anchor are also localized in the terminal region, electrostatic repulsion between anchoring units of PGM molecules on a nonpolar PDMS surface leads to weakening of the mechanical integrity of the films.
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| 8. |
- Madsen, Mikkel, et al.
(författare)
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Unaided efficient transglutaminase cross-linking of whey proteins strongly impacts the formation and structure of protein alginate particles
- 2022
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Ingår i: Food Chemistry: Molecular Sciences. - : Elsevier BV. - 2666-5662. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- There is a dogma within whey protein modification, which dictates the necessity of pretreatment to enzymatic cross-linking of β-lactoglobulin (β-Lg). Here microbial transglutaminase (MTG) cross-linked whey proteins and β-Lg effectively in 50 mM NaHCO3, pH 8.5, without pretreatment. Cross-linked β-Lg spanned 18 to >240 kDa, where 6 of 9 glutamines reacted with 8 of 15 lysines. The initial isopeptide bond formation caused loss of β-Lg native structure with t1/2 = 3 h, while the polymerization occurred with t1/2 = 10 h. Further, cross-linking effects on protein carbohydrate interaction have been overlooked, leaving a gap in understanding of these complex food matrices. Complexation with alginate showed that β-Lg cross-linking decreased onset of particle formation, hydrodynamic diameter, stoichiometry (β-Lg/alginate) and dissociation constant. The complexation was favored at higher temperatures (40 °C), suggesting that hydrophobic interactions were important. Thus, β-Lg was cross-linked without pretreatment and the resulting polymers gave rise to altered complexation with alginate.
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| 9. |
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| 10. |
- Møller, Marie Sofie, et al.
(författare)
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Structural biology of starch-degrading enzymes and their regulation
- 2016
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Ingår i: Current Opinion in Structural Biology. - : Elsevier BV. - 0959-440X. ; 40, s. 33-42
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Forskningsöversikt (refereegranskat)abstract
- Starch is a major energy source for all domains of life. Recent advances in structures of starch-degrading enzymes encompass the substrate complex of starch debranching enzyme, the function of surface binding sites in plant isoamylase, details on individual steps in the mechanism of plant disproportionating enzyme and a self-stabilised conformation of amylose accommodated in the active site of plant α-glucosidase. Important inhibitor complexes include a flavonol glycoside, montbretin A, binding at the active site of human pancreatic α-amylase and barley limit dextrinase inhibitor binding to the debranching enzyme, limit dextrinase using a new binding mode for cereal protein inhibitors.
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| 11. |
- Møller, Marie Sofie, et al.
(författare)
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Structure and function of α-glucan debranching enzymes
- 2016
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Ingår i: Cellular and Molecular Life Sciences. - : Springer Science and Business Media LLC. - 1420-682X .- 1420-9071. ; 73:14, s. 2619-2641
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Forskningsöversikt (refereegranskat)abstract
- α-Glucan debranching enzymes hydrolyse α-1,6-linkages in starch/glycogen, thereby, playing a central role in energy metabolism in all living organisms. They belong to glycoside hydrolase families GH13 and GH57 and several of these enzymes are industrially important. Nine GH13 subfamilies include α-glucan debranching enzymes; isoamylase and glycogen debranching enzymes (GH13_11); pullulanase type I/limit dextrinase (GH13_12–14); pullulan hydrolase (GH13_20); bifunctional glycogen debranching enzyme (GH13_25); oligo-1 and glucan-1,6-α-glucosidases (GH13_31); pullulanase type II (GH13_39); and α-amylase domains (GH13_41) in two-domain amylase–pullulanases. GH57 harbours type II pullulanases. Specificity differences, domain organisation, carbohydrate binding modules, sequence motifs, three-dimensional structures and specificity determinants are discussed. The phylogenetic analysis indicated that GH13_39 enzymes could represent a “missing link” between the strictly α-1,6-specific debranching enzymes and the enzymes with dual specificity and α-1,4-linkage preference.
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| 12. |
- Teze, David, et al.
(författare)
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Rational Enzyme Design without Structural Knowledge : A Sequence-Based Approach for Efficient Generation of Transglycosylases
- 2021
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Ingår i: Chemistry: A European Journal. - : Wiley. - 1521-3765 .- 0947-6539. ; 27:40, s. 10323-10334
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Tidskriftsartikel (refereegranskat)abstract
- Glycobiology is dogged by the relative scarcity of synthetic, defined oligosaccharides. Enzyme-catalysed glycosylation using glycoside hydrolases is feasible but is hampered by the innate hydrolytic activity of these enzymes. Protein engineering is useful to remedy this, but it usually requires prior structural knowledge of the target enzyme, and/or relies on extensive, time-consuming screening and analysis. Here we describe a straightforward strategy that involves rational rapid in silico analysis of protein sequences. The method pinpoints 6-12 single mutant candidates to improve transglycosylation yields. Requiring very little prior knowledge of the target enzyme other than its sequence, the method is generic and procures catalysts for the formation of glycosidic bonds involving various d / l -, α/β-pyranosides or furanosides, and exo - and endo -action. Moreover, mutations validated in one enzyme can be transposed to others, even distantly related enzymes.
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