| 1. |
- Ahlgren, Eva Christina, et al.
(författare)
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Iron-induced oligomerization of human FXN81-210 and bacterial CyaY frataxin and the effect of iron chelators
- 2017
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:12
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Tidskriftsartikel (refereegranskat)abstract
- Patients suffering from the progressive neurodegenerative disease Friedreich’s ataxia have reduced expression levels of the protein frataxin. Three major isoforms of human frataxin have been identified, FXN42-210, FXN56-210 and FXN81-210, of which FXN81-210 is considered to be the mature form. Both long forms, FXN42-210 and FXN56-210, have been shown to spontaneously form oligomeric particles stabilized by the extended N-terminal sequence. The short variant FXN81-210, on other hand, has only been observed in the monomeric state. However, a highly homologous E. coli frataxin CyaY, which also lacks an N-terminal extension, has been shown to oligomerize in the presence of iron. To explore the mechanisms of stabilization of short variant frataxin oligomers we compare here the effect of iron on the oligomerization of CyaY and FXN81-210. Using dynamic light scattering, small-angle X-ray scattering, electron microscopy (EM) and cross linking mass spectrometry (MS), we show that at aerobic conditions in the presence of iron both FXN81-210 and CyaY form oligomers. However, while CyaY oligomers are stable over time, FXN81-210 oligomers are unstable and dissociate into monomers after about 24 h. EM and MS studies suggest that within the oligomers FXN81-210 and CyaY monomers are packed in a head-to-tail fashion in ring-shaped structures with potential iron-binding sites located at the interface between monomers. The higher stability of CyaY oligomers can be explained by a higher number of acidic residues at the interface between monomers, which may result in a more stable iron binding. We also show that CyaY oligomers may be dissociated by ferric iron chelators deferiprone and DFO, as well as by the ferrous iron chelator BIPY. Surprisingly, deferiprone and DFO stimulate FXN81-210 oligomerization, while BIPY does not show any effect on oligomerization in this case. The results suggest that FXN81-210 oligomerization is primarily driven by ferric iron, while both ferric and ferrous iron participate in CyaY oligomer stabilization. Analysis of the amino acid sequences of bacterial and eukaryotic frataxins suggests that variations in the position of the acidic residues in helix 1, β-strand 1 and the loop between them may control the mode of frataxin oligomerization.
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| 2. |
- Bhattacharya, Abhishek, et al.
(författare)
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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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Ingår i: Microorganisms. - : MDPI AG. - 2076-2607. ; 10:12
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Tidskriftsartikel (refereegranskat)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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| 3. |
- Bhattacharya, Abhishek, et al.
(författare)
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β-Mannanase BoMan26B from Bacteroides ovatus produces mannan-oligosaccharides with prebiotic potential from galactomannan and softwood β-mannans
- 2021
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Ingår i: LWT. - : Elsevier BV. - 0023-6438. ; 151
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Tidskriftsartikel (refereegranskat)abstract
- Galactomannan (GM) in legumes and acetyl-galactoglucomannan (AcGGM) in softwoods are wide-spread β-mannans. Their depolymerisation is catalyzed by β-mannanases. We have investigated a cell-surface exposed and galactose-tolerant β-mannanase (BoMan26B) from the abundant gut bacterium Bacteroides ovatus. Glycosidases from the gut microbiota have potential for production of prebiotics, such as dietary saccharides that would promote beneficial bacteria in the gut. BoMan26B was explored for production of potential prebiotics. Using the above β-mannans as substrate we investigated the product profiles using a herein developed new high-resolution anion-exchange chromatography procedure. The produced linear and galactosyl-decorated β-mannan-oligosaccharides (MOS/GMOS) were mainly of degree of polymerization (DP) 2–6, consistent with the glycan-binding subsites of BoMan26B. Some GM and AcGGM products were acetylated. DP 2–6 MOS were produced at a yield of 30 and 33% (w/w) from GM and AcGGM, respectively. In addition, about as much DP 2–6 GMOS were produced, assessed using guar α-galactosidase as analytical aid. Growth studies using the human gut bacteria Bifidobacterium adolescentis ATCC 15703 (acetate producer) and Roseburia hominis DSMZ 6839 (butyrate producer) revealed significant differences in utilization of specific MOS/GMOS. The prebiotic potential of the MOS/GMOS generated by BoMan26B was further underlined by the observation that both bacterial strains produced short-chain fatty acids.
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| 4. |
- Birgersson, Simon, et al.
(författare)
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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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Ingår i: Catalysts. - 2073-4344. ; 13:9
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Tidskriftsartikel (refereegranskat)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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| 5. |
- Butler, Samuel J., et al.
(författare)
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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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Ingår i: Process Biochemistry. - : Elsevier BV. - 1359-5113. ; 112, s. 154-166
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Tidskriftsartikel (refereegranskat)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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| 6. |
- Bågenholm, Viktoria, et al.
(författare)
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A surface-exposed GH26 -mannanase from Bacteroides ovatus : Structure, role, and phylogenetic analysis of BoMan26B
- 2019
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 294:23, s. 9100-9117
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Tidskriftsartikel (refereegranskat)abstract
- The galactomannan utilization locus (BoManPUL) of the human gut bacterium Bacteroides ovatus encodes BoMan26B, a cell-surface– exposed endomannanase whose functional and structural features have been unclear. Our study now places BoMan26B in context with related enzymes and reveals the structural basis for its specificity. BoMan26B prefers longer substrates and is less restricted by galactose side-groups than the mannanase BoMan26A of the same locus. Using galactomannan, BoMan26B generated a mixture of (galactosyl) manno-oligosaccharides shorter than mannohexaose. Three defined manno-oligosaccharides had affinity for the SusD-like surface–exposed glycan-binding protein, predicted to be implicated in saccharide transport. Co-incubation of BoMan26B and the periplasmic -galactosidase BoGal36A increased the rate of galactose release by about 10-fold compared with the rate without BoMan26B. The results suggested that BoMan26B performs the initial attack on galactomannan, generating oligosaccharides that after transport to the periplasm are processed by BoGal36A. A crystal structure of BoMan26B with galactosyl-mannotetraose bound in subsites 5 to 2 revealed an open and long active-site cleft with Trp-112 in subsite 5 concluded to be involved in mannosyl interaction. Moreover, Lys-149 in the 4 subsite interacted with the galactosyl side-group of the ligand. A phylogenetic tree consisting of GH26 enzymes revealed four strictly conserved GH26 residues and disclosed that BoMan26A and BoMan26B reside on two distinct phylogenetic branches (A and B). The three other branches contain lichenases, xylanases, or enzymes with unknown activities. Lys-149 is conserved in a narrow part of branch B, and Trp-112 is conserved in a wider group within branch B.
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| 7. |
- Dujon, B, et al.
(författare)
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The nucleotide sequence of Saccharomyces cerevisiae chromosome XV
- 1997
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 387:6632, s. 98-102
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Tidskriftsartikel (refereegranskat)abstract
- Chromosome XV was one of the last two chromosomes of Saccharomyces cerevisiae to be discovered(1). It is the third-largest yeast chromosome after chromosomes XII and IV, and is very similar in size to chromosome VII. It alone represents 9% of the yeast genome (8% if ribosomal DNA is included). When systematic sequencing of chromosome XV was started, 93 genes or markers were identified, and most of them were mapped(2). However, very little else was known about chromosome XV which, in contrast to shorter chromosomes, had not been the object of comprehensive genetic or molecular analysis. It was therefore decided to start sequencing chromosome XV only in the third phase of the European Yeast Genome Sequencing Programme, after experience was gained on chromosomes III, XI and II (refs 3-5). The sequence of chromosome XV has been determined from a set of partly overlapping cosmid clones derived from a unique yeast strain, and physically mapped at 3.3-kilobase resolution before sequencing. As well as numerous new open reading frames (ORFs) and genes encoding tRNA or small RNA molecules, the sequence of 1,091,283 base pairs confirms the high proportion of orphan genes and reveals a number of ancestral and successive duplications with other yeast chromosomes.
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| 8. |
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| 9. |
- 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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| 10. |
- von Freiesleben, Pernille, et al.
(författare)
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Crystal structure and substrate interactions of an unusual fungal non-CBM carrying GH26 endo-β-mannanase from Yunnania penicillata
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Endo-β(1 → 4)-mannanases (endomannanases) catalyse degradation of β-mannans, an abundant class of plant polysaccharides. This study investigates structural features and substrate binding of YpenMan26A, a non-CBM carrying endomannanase from Yunnania penicillata. Structural and sequence comparisons to other fungal family GH26 endomannanases showed high sequence similarities and conserved binding residues, indicating that fungal GH26 endomannanases accommodate galactopyranosyl units in the −3 and −2 subsites. Two striking amino acid differences in the active site were found when the YpenMan26A structure was compared to a homology model of Wsp.Man26A from Westerdykella sp. and the sequences of nine other fungal GH26 endomannanases. Two YpenMan26A mutants, W110H and D37T, inspired by differences observed in Wsp.Man26A, produced a shift in how mannopentaose bound across the active site cleft and a decreased affinity for galactose in the −2 subsite, respectively, compared to YpenMan26A. YpenMan26A was moreover found to have a flexible surface loop in the position where PansMan26A from Podospora anserina has an α-helix (α9) which interacts with its family 35 CBM. Sequence alignment inferred that the core structure of fungal GH26 endomannanases differ depending on the natural presence of this type of CBM. These new findings have implications for selecting and optimising these enzymes for galactomannandegradation.
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