| 1. |
- Bhattacharya, Abhishek, et al.
(author)
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Cross-Feeding and Enzymatic Catabolism for Mannan-Oligosaccharide Utilization by the Butyrate-Producing Gut Bacterium Roseburia hominis A2-183
- 2022
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In: Microorganisms. - : MDPI AG. - 2076-2607. ; 10:12
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Journal article (peer-reviewed)abstract
- β-Mannan is abundant in the human diet and in hemicellulose derived from softwood. Linear or galactose-substituted β-mannan-oligosaccharides (MOS/GMOSs) derived from β-mannan are considered emerging prebiotics that could stimulate health-associated gut microbiota. However, the underlying mechanisms are not yet resolved. Therefore, this study investigated the cross-feeding and metabolic interactions between Bifidobacterium adolescentis ATCC 15703, an acetate producer, and Roseburia hominis A2-183 DSMZ 16839, a butyrate producer, during utilization of MOS/GMOSs. Cocultivation studies suggest that both strains coexist due to differential MOS/GMOS utilization, along with the cross-feeding of acetate from B. adolescentis E194a to R. hominis A2-183. The data suggest that R. hominis A2-183 efficiently utilizes MOS/GMOS in mono- and cocultivation. Notably, we observed the transcriptional upregulation of certain genes within a dedicated MOS/GMOS utilization locus (RhMosUL), and an exo-oligomannosidase (RhMan113A) gene located distally in the R. hominis A2-183 genome. Significantly, biochemical analysis of β-1,4 mannan-oligosaccharide phosphorylase (RhMOP130A), α-galactosidase (RhGal36A), and exo-oligomannosidase (RhMan113A) suggested their potential synergistic role in the initial utilization of MOS/GMOSs. Thus, our results enhance the understanding of MOS/GMOS utilization by potential health-promoting human gut microbiota and highlight the role of cross-feeding and metabolic interactions between two secondary mannan degraders inhabiting the same ecological niche in the gut.
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| 2. |
- Birgersson, Simon
(author)
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Exploring beta-mannan acting glycosidase families in hydrolysis and mannosyl transfer
- 2024
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Doctoral thesis (other academic/artistic)abstract
- The thesis revolves around the study of glycoside hydrolases (GH), specifically GH5 β-mannanases and GH36 α-galactosidase (I-III), as well as a GH130 mannoside phosphorylase (I, IV). These enzymes are investigated for their actions on and conversion of hemicellulosic β-mannan, including food-grade galactomannan and softwood galactoglucomannan, along with manno-oligosaccharides (MOS) derived from such polymers. The focus lies on exploring the transferase activity, specifically transglycosylation, of retaining GHs, elucidating the molecular and structural mechanisms governing these reactions, and assessing their potential applications in synthesizing novel glycosides like MOS and allyl glycosides from renewable glycans (II, III).Paper I explores the utilization of β-mannan-oligosaccharides (MOS/GMOSs) by Roseburia hominis, and its interactions with Bifidobacterium adolescentis, revealing differential utilization patterns, cross-feeding of acetate, and potential synergistic roles of specific enzymes shown to be upregulated in the presence of MOS/GMOS by R. hominis. In Paper II, A novel double mutant (R171K/E205D) of the catalytic module (CM) of the fam- ily GH5 Trichoderma reesei β-mannanase (TrMan5A) showed enhanced transglycosylation capacity, particularly with mannotetraose and allyl alcohol, surpassing wild-type TrMan5A and offering potential applications in the enzymatic synthesis of novel biomaterials and glycopolymers through allyl glycoside production. The +2 subsite of MeMan5A, a GH5 β-mannanase from the blue mussel, was studied by substituting two tryptophans with alanines in Paper III. These substitutions impaired MeMan5A’s hydrolytic activity, affecting transglycosylation with saccha- rides but not alcoholysis. In Paper IV, the mannan oligosaccharide phosphorylase RhMOP130A, first investigated in Paper I was investigated in terms of its structure and studied for its synthetic potential in the reverse phospho- rlysis reaction direction, allowing for elongation of mannan oligosaccharide using activated mannose phosphate. RhMOP130A showed the ability to elongate allyl mannose, such as produced in Paper II, making it an interesting tool for modification of novel glycosides.The findings of this thesis enhance our comprehension of the molecular factors and reaction parameters that impact the effective conversion of β-mannans into novel glycosides. This showcases the viability of utilizing β- mannanases in the enzymatic synthesis of novel bio-materials.
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| 3. |
- Birgersson, Simon, et al.
(author)
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Flexibility and Function of Distal Substrate-Binding Tryptophans in the Blue Mussel β-Mannanase MeMan5A and Their Role in Hydrolysis and Transglycosylation
- 2023
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In: Catalysts. - 2073-4344. ; 13:9
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Journal article (peer-reviewed)abstract
- β-Mannanases hydrolyze β-mannans, important components of plant and microalgae cell walls. Retaining β-mannanases can also catalyze transglycosylation, forming new β-mannosidic bonds that are applicable for synthesis. This study focused on the blue mussel (Mytilus edulis) GH5_10 β-mannanase MeMan5A, which contains two semi-conserved tryptophans (W240 and W281) in the distal subsite +2 of its active site cleft. Variants of MeMan5A were generated by replacing one or both tryptophans with alanines. The substitutions reduced the enzyme’s catalytic efficiency (kcat/Km using galactomannan) by three-fold (W281A), five-fold (W240A), or 20-fold (W240A/W281A). Productive binding modes were analyzed by 18O labeling of hydrolysis products and mass spectrometry. Results show that the substitution of both tryptophans was required to shift away from the dominant binding mode of mannopentaose (spanning subsites −3 to +2), suggesting that both tryptophans contribute to glycan binding. NMR spectroscopy and molecular dynamics simulations were conducted to analyze protein flexibility and glycan binding. We suggest that W240 is rigid and contributes to +2 subsite mannosyl specificity, while W281 is flexible, which enables stacking interactions in the +2 subsite by loop movement to facilitate binding. The substitutions significantly reduced or eliminated transglycosylation with saccharides as glycosyl acceptors but had no significant effect on reactions with alcohols.
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| 4. |
- Butler, Samuel J., et al.
(author)
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Transglycosylation by β-mannanase TrMan5A variants and enzyme synergy for synthesis of allyl glycosides from galactomannan
- 2022
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In: Process Biochemistry. - : Elsevier BV. - 1359-5113. ; 112, s. 154-166
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Journal article (peer-reviewed)abstract
- Retaining β-mannanases are glycoside hydrolases (GHs) that can potentially be applied for synthesis of glycosides by catalysis of transglycosylation reactions. A novel active-site double mutant (R171K/E205D) of the catalytic module (CM) of the family GH5 Trichoderma reesei β-mannanase (TrMan5A) was expressed in Pichia pastoris and purified. TrMan5A, CM and CM-variants R171K and R171K/E205D had pH optima between pH 4.0–5.3 and showed >80 % remaining activity after incubation at 40 °C for 48 h. The enzymes were screened for transglycosylation capacity toward oligomeric and polymeric donor substrates and alcohol acceptors using mass-spectrometry. Hydrolysis and transglycosylation products were analysed by a novel HPLC procedure using an NH2 column. R171K/E205D was superior in reactions with mannotetraose and the acceptor allyl alcohol, it had twice as high propensity for transglycosylation as wild-type TrMan5A. Wild-type TrMan5A produced the highest amounts of allyl β-mannosides (with 1–3 mannosyls) from locust bean galactomannan. Applying enzyme synergy, adding the GH27 guar α-galactosidase to the reaction (to cleave off galactomannan side-groups), gave a 2.1-fold increase of allyl mannosides and simultaneously a significant production of allyl galactopyranoside, increasing overall yield of allyl glycosides 4.4-fold, from 2.2% to 9.8%. The enzymatic synthesis of reactive allyl glycosides opens up for production of novel biomaterials and glycopolymers.
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| 5. |
- Wernersson, Sven, et al.
(author)
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Cosolvent Dimethyl Sulfoxide Influences Protein-Ligand Binding Kinetics via Solvent Viscosity Effects : Revealing the Success Rate of Complex Formation Following Diffusive Protein-Ligand Encounter
- 2023
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In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 62:1, s. 44-52
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Journal article (peer-reviewed)abstract
- Protein-ligand-exchange kinetics determines the duration of biochemical signals and consequently plays an important role in drug design. Binding studies commonly require solubilization of designed ligands in solvents such as dimethyl sulfoxide (DMSO), resulting in residual amounts of DMSO following titration of solubilized ligands into aqueous protein samples. Therefore, it is critical to establish whether DMSO influences protein-ligand binding. Here, we address the general and indirect effect of DMSO on protein-ligand binding caused by solvent viscosity, which is strongly dependent on the relative concentrations of DMSO and water. As a model system, we studied the binding of a drug-like ligand to the carbohydrate recognition domain of galectin-3 in the presence of variable amounts of DMSO. We used isothermal titration calorimetry to characterize binding thermodynamics and 15N NMR relaxation to monitor kinetics. The binding enthalpy is not affected, but we observe a subtle trend of increasingly unfavorable entropy of binding, and consequently decreased affinity, with increasing DMSO concentration. The increasing concentration of DMSO results in a reduced association rate of binding, while the dissociation rate is less affected. The observed association rate is inversely proportional to the viscosity of the DMSO-water mixture, as expected from theory, but significantly reduced from the diffusion-controlled limit. By comparing the viscosity dependence of the observed association rate with that of the theoretical diffusion-controlled association rate, we estimate the success rate of productive complex formation following an initial encounter of proteins and ligands, showing that only one out of several hundred binding “attempts” are successful.
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