| 1. |
- Becker, D., et al.
(författare)
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Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum : the pH behaviour of Trichoderma reesei CeI7A and its E223S/A224H/L225V/T226A/D262G mutant
- 2001
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 356, s. 19-30
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Tidskriftsartikel (refereegranskat)abstract
- The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala(224)) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves, The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 Angstrom (= 0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 Angstrom contact between N-2 and O'(1). The pH variation of k(cat)/K-m for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wildtype and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) shifting from 5.99+/-0.02 to 6.78+/-0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K-m values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) values for cellobiosides are higher for the mutant. This we attribute to reduced nonproductive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.
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| 2. |
- Woestenenk, Esmeralda A., et al.
(författare)
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The solution structure of ribosomal protein L18 from Thermus thermophilus reveals a conserved RNA-binding fold
- 2002
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 363:3, s. 553-561
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Tidskriftsartikel (refereegranskat)abstract
- We have determined the solution structure of ribosomal protein L18 from Thermus thermophilus. L18 is a 12.5 kDa protein of the large subunit of the ribosome and binds to both 5 S and 23 S rRNA. In the uncomplexed state L18 folds to a mixed α/β globular structure with a long disordered N-terminal region. We compared our high-resolution structure with RNA-complexed L 18 from Haloarcula marismortui and T. thermophilus to examine RNA-induced as well as species-dependent structural differences. We also identified T. thermophilus S11 as a structural homologue and found that the structures of the RNA-recognition sites are conserved. Important features, for instance a bulge in the RNA-contacting β-sheet, are conserved in both proteins. We suggest that the L18 fold recognizes a specific RNA motif and that the resulting RNA-protein-recognition module is tolerant to variations in sequence.
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| 3. |
- Bessueille, Laurence, et al.
(författare)
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Plasma membrane microdomains from hybrid aspen cells are involved in cell wall polysaccharide biosynthesis
- 2009
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 420, s. 93-103
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Tidskriftsartikel (refereegranskat)abstract
- Detergent-resistant plasma membrane microdomains [DRMs (detergent-resistant membranes)] were isolated recently from several plant species. As for animal cells, a large range of cellular functions, such as signal transduction, endocytosis and protein trafficking, have been attributed to plant lipid rafts and DRMs. The data available are essentially based on protcomics and more approaches need to be undertaken to elucidate the precise function of individual populations of DRMs in plants. We report here the first isolation of DRMs from purified plasma membranes of a tree species, the hybrid aspen Populus tremula x tremuloides, and their biochemical characterization. Plasma membranes were solubilized with Triton X-100 and the resulting DRMs were isolated by flotation in sucrose density gradients. The DRMs were enriched in sterols, sphingolipids and glycosylphosphatidylinositol-anchored proteins and thus exhibited similar properties to DRMs from other species. However, they contained key carbohydrate synthases involved in cell wall polysaccharide biosynthesis, namely callose [(1 -> 3)-beta-D-glucan] and cellulose synthases. The association of these enzymes with DRMs was demonstrated using specific glucan synthase assays and antibodies, as well as biochemical and chemical approaches for the characterization of the polysaccharides synthesized in vitro by the isolated DRMs. More than 70% of the total glucan synthase activities present in the original plasma membranes was associated with the DRM fraction. In addition to shedding light on the lipid environment of callose and cellulose synthases, our results demonstrate the involvement of DRMs in the biosynthesis of important cell wall polysaccharides. This novel concept suggests a function of plant membrane microdomains in cell growth and morphogenesis.
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| 4. |
- Faijes, M., et al.
(författare)
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In vitro synthesis of a crystalline (1 -> 3,1 -> 4)-beta-D-glucan by a mutated (1 -> 3,1 -> 4)-beta-D-glucanase from Bacillus
- 2004
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 380, s. 635-641
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Tidskriftsartikel (refereegranskat)abstract
- Oligo- and poly-saccharides have a large number of important biological functions, and they occur in natural composite materials, such as plant cell walls, where they self-assemble during biosynthesis in a poorly understood manner. They can also be used for the formation of artificial composite materials with industrial applications. Fundamental and applied research in biology and nanobiotechnology would benefit from the possibility of synthesizing tailor-made oligo-/poly-saccharides. In the present paper, we demonstrate that such syntheses are possible using genetically modified glycoside hydrolases, i.e. glycosynthases. The ability of the endoglycosynthase derived from Bacillus ( 1 --> 3,1 --> 4)-beta-D-glucanase to catalyse self-condensation of sugar donors was exploited for the in vitro synthesis of a regular polysaccharicle. The specificity of the enzyme allowed the polymerization of alpha-laminaribiosyl fluoride via the formation of (1 --> 4)-beta-linkages to yield a new linear crystalline (1 --> 3,1 --> 4)-beta-D-glucan with a repeating 4betaG3betaG unit. MS and methylation analyses indicated that the in vitro product consisted of a mixture of oligosaccharides, the one having a degree of polymerization of 12 being the most abundant. Morphological characterization revealed that the (1 --> 3,1 --> 4)-beta-D-glucan forms spherulites which are composed of platelet crystals. X-ray and electron diffraction analyses allowed the proposition of a putative crystallographic structure which corresponds to a monoclinic unit cell with a = 0.834 nm, b = 0.825 nm, c = 2.04 nm and gamma = 90.5degrees. The dimensions of the ab plane are similar to those of cellulose I-beta, but the length of the c-axis is nearly twice that of cellulose I. It is proposed that four glucose residues are present in an extended conformation along the c-axis of the unit cell. The data presented show that glycosynthases represent promising enzymic systems for the synthesis of novel polysaccharides with specific and controlled structures, and for the analysis in vitro of the mechanisms of polymerization and crystallization of potysaccharides.
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| 5. |
- Henriksson, G, et al.
(författare)
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Studies of cellulose binding by cellobiose dehydrogenase and a comparison with cellobiohydrolase 1
- 1997
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 324, s. 833-838
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Tidskriftsartikel (refereegranskat)abstract
- The binding isotherm to cellulose of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium has been compared with that of cellobiohydrolase 1 (CBH 1) from Trichoderma reesei. CDH binds more strongly but more sparsely to cellulose than does CBH 1. In a classical Scatchard analysis, a better fit to a one-site binding model was obtained for CDH than for CBH 1. The binding of both enzymes decreased in the presence of ethylene glycol, increased in the presence of ammonium sulphate and was unaffected by sodium chloride. Attempts to localize the cellulose-binding site on CDH have also been made by exposing enzymically digested CDH to cellulose and isolating the cellulose-bound peptides. The results suggest that the cellulose-binding site is located internally in the amino acid sequence of CDH.
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| 6. |
- Henriksson, H., et al.
(författare)
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N-linked glycosylation of native and recombinant cauliflower xyloglucan endotransglycosylase 16A
- 2003
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 375, s. 61-73
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Tidskriftsartikel (refereegranskat)abstract
- The gene encoding a XET (xyloglucan endotransglycosylase) from cauliflower (Brassica oleracea var. botrytis) florets has been cloned and sequenced. Sequence analysis indicated a high degree of similarity to other XET enzymes belonging to glycosyl hydrolase family 16 (GH16). In addition to the conserved GH16 catalytic sequence motif EIDFE, there exists one potential N-linked glycosylation site. which is also highly conserved in XET enzymes from this family. Purification of the corresponding protein from extracts of cauliflower florets allowed the fractionation of a single, pure glycoform. which was analysed by MS techniques. Accurate protein mass determination following the enzymic deglycosylation of this glycoform indicated the presence of a high-mannose-type glycan of the general structure GlcNAc(2)Man(6). LC/MS and MS/MS (tandem MS) analysis provided supporting evidence for this structure and confirmed that the glycosylation site (underlined) was situated close to the predicted catalytic residues in the conserved sequence YLSSTNNEHDEIDFEFLGNRTGQPVILQTNVFTGGK. Heterologous expression in Pichia pastoris produced a range of protein glycoforms, which were, on average, more highly mannosylated than the purified native enzyme. This difference in glycosylation did not influence the apparent enzymic activity of the enzyme significantly. However, the removal of high-mannose glycosylation in recombinant cauliflower XET by endoglycosidase H, quantified by electrospray-ionization MS, caused a 40 % decrease in the transglycosylation activity of the enzyme. No hydrolytic activity was detected in native or heterologously expressed BobXET16A, even when almost completely deglycosylated.
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| 7. |
- Knorpp, Carina, et al.
(författare)
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Evidence for a novel ATP-dependent membrane-associated protease in spinach leaf mitochondria.
- 1995
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 310 ( Pt 2)
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Tidskriftsartikel (refereegranskat)abstract
- We report the presence of an ATP-dependent proteolytic activity in spinach (Spinacia oleracea) leaf mitochondria. The proteolysis was observed as degradation of newly imported precursor protein. The precursor studied was that of the ATP synthase F1 beta subunit of Nicotiana plumbaginifolia, transcribed and translated in vitro. Degradation of pre-F1 beta was observed during kinetic studies of import in vitro. The degradation was characterized in chase experiments in which the precursor was imported into mitochondria. The import reaction was subsequently stopped by the addition of valinomycin and oligomycin. The fate of the imported precursor inside the mitochondria was monitored under different experimental conditions. There was no proteolytic degradation of the newly imported precursor at 15 degrees C, whereas 50% of the precursor was degraded after a 45 min incubation at 25 degrees C. The proteolytic activity was found to be ATP-dependent and was partially inhibited by a metal chelator, o-phenanthroline. Fractionation of mitochondria prior to degradation showed that all the ATP-dependent degradative activity was associated with the mitochondrial membrane fraction. The membrane-bound protease was inhibited by Pefabloc [4-(2-aminoethyl)-benzenesulphonyl fluoride hypochloride], an inhibitor of serine-type proteases and by N-ethylmaleimide, a thiol group reagent. Our studies thus describe a novel ATP-dependent membrane-associated serine-type protease in plant mitochondria that is capable of degrading newly imported non-assembled proteins.
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| 8. |
- Larsbrink, Johan, et al.
(författare)
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Structural and enzymatic characterization of a glycoside hydrolase family 31 alpha-xylosidase from Cellvibrio japonicus involved in xyloglucan saccharification
- 2011
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 436, s. 567-580
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Tidskriftsartikel (refereegranskat)abstract
- The desire for improved methods of biomass conversion into fuels and feedstocks has re-awakened interest in the enzymology of plant cell wall degradation. The complex polysaccharide xyloglucan is abundant in plant matter, where it may account for up to 20% of the total primary cell wall carbohydrates. Despite this, few studies have focused on xyloglucan saccharification, which requires a consortium of enzymes including endo-xyloglucanases, alpha-xylosidases, beta-galactosidases and alpha-L-fucosidases, among others. In the present paper, we show the characterization of Xy131A, a key alpha-xylosidase in xyloglucan utilization by the model Gram-negative soil saprophyte Cellvibrio japonicus. CjXy131A exhibits high regiospecificity for the hydrolysis of XGOs (xylogluco-oligosaccharides), with a particular preference for longer substrates. Crystallographic structures of both the apo enzyme and the trapped covalent 5-fluoro-beta-xylosyl-enzyme intermediate, together with docking studies with the XXXG heptasaccharide, revealed, for the first time in GH31 (glycoside hydrolase family 31), the importance of PA14 domain insert in the recognition of longer oligosaccharides by extension of the active-site pocket. The observation that CjXy131A was localized to the outer membrane provided support for a biological model of xyloglucan utilization by C. japonicas, in which XGOs generated by the action of a secreted endo-xyloglucanase are ultimately degraded in close proximity to the cell surface. Moreover, the present study diversifies the toolbox of glycosidases for the specific modification and saccharification of cell wall polymers for biotechnological applications.
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| 9. |
- Lombard, Vincent, et al.
(författare)
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A hierarchical classification of polysaccharide lyases for glycogenomics
- 2010
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 432, s. 437-444
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Tidskriftsartikel (refereegranskat)abstract
- Carbohydrate-active enzymes face huge substrate diversity in a highly selective manner using only a limited number of available folds. They are therefore subjected to multiple divergent and convergent evolutionary events. This and their frequent modularity render their functional annotation in genomes difficult in a number of cases. In the present paper, a classification of polysaccharide lyases (the enzymes that cleave polysaccharides using an elimination instead of a hydrolytic mechanism) is shown thoroughly for the first time. Based on the analysis of a large panel of experimentally characterized polysaccharide lyases, we examined the correlation of various enzyme properties with the three levels of the classification: fold, family and subfamily. The resulting hierarchical classification, which should help annotate relevant genes in genomic efforts, is available and constantly updated at the Carbohydrate-Active Enzymes Database (http://www.cazy.org).
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| 10. |
- Mackenzie, L F, et al.
(författare)
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Crystal structure of the family 7 endoglucanase I (Cel7B) from Humicola insolens at 2.2 angstrom resolution and identification of the catalytic nucleophile by trapping of the covalent glycosyl-enzyme intermediate
- 1998
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 335, s. 409-416
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Tidskriftsartikel (refereegranskat)abstract
- Cellulose is the major polysaccharide component of the plant cell wall and the most abundant naturally produced macromolecule on Earth. The enzymic degradation of cellulose, by cellulases, is therefore of great environmental and commercial significance. Cellulases are found in 12 of the glycoside hydrolase families classified according to their amino acid sequence similarities. Endoglucanase I (Cel7B), from the soft-rot fungus Humicola insolens, is a family 7 enzyme. The structure of the native form of Cel7B from H. insolens at 2.2 Å resolution has been solved by molecular replacement using the known Trichoderma reesei cellobiohydrolase I [Divne, Ståhlberg, Reinikainen, Ruohonen, Pettersson, Knowles, Teeri and Jones (1994) Science265, 524–528] structure as the search model. Cel7B catalyses hydrolysis of the β-1,4 glycosidic linkages in cellulose with net retention of anomeric configuration. The catalytic nucleophile at the active site of Cel7B has been identified as Glu-197 by trapping of a 2-deoxy-2-fluorocellotriosyl enzyme intermediate and identification of the labelled peptide in peptic digests by tandem MS. Site-directed mutagenesis of both Glu-197 and the prospective catalytic acid, Glu-202, results in inactive enzyme, confirming the critical role of these groups for catalysis.
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