| 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. |
- 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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| 3. |
- Kallas, Åsa M., et al.
(författare)
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Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremula x tremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris
- 2005
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Ingår i: Biochemical Journal. - 0264-6021. ; 390, s. 105-113
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Tidskriftsartikel (refereegranskat)abstract
- The cDNA encoding a xyloglucan endotransglycosylase, PttXET16A, from hybrid aspen (Populus tremula x tremuloides) has been isolated from an expressed sequence tag library and expressed in the methylotrophic yeast Pichia pastoris. Sequence analysis indicated a high degree of similarity with other proteins in the XTH (xyloglucan transglycosylase/hydrolase) gene subfamily of GH16 (glycoside hydrolase family 16). In addition to the conserved GH16 catalytic sequence motif, PttXET16A contains a conserved N-glycosylation site situated proximal to the predicted catalytic residues. MS analysis indicated that the recombinant PttXET16A expressed in P. pastoris is heterogeneous due to the presence of variable N-glycosylation and incomplete cleavage of the a-factor secretion signal peptide. Removal of the N-glycan by endoglycosidase H treatment did not influence the catalytic activity significantly. Similarly, site-directed mutagenesis of Asn(93) to serine to remove the N-glycosylation site resulted in an enzyme which was comparable with the wild-type enzyme in specific activity and thermal stability but had clearly reduced solubility. Hydrolytic activity was detected neither in wild-type PttXET16A before or after enzymatic deglycosylation nor in PttXET16A N93S (Asn(93) -> Ser) mutant.
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| 4. |
- 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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| 5. |
- 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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| 6. |
- Saura-Valls, M., et al.
(författare)
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Kinetic analysis using low-molecular mass xyloglucan oligosaccharides defines the catalytic mechanism of a Populus xyloglucan endotransglycosylase
- 2006
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 395, s. 99-106
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Tidskriftsartikel (refereegranskat)abstract
- Plant XETs [XG (xyloglucan) endotransglycosylases] catalyse the transglycosylation front a XG donor to a XG or low-molecular-mass XG fragment Lis the acceptor, and are thought to be important enzymes in the formation and remodelling of the cellulose-XG three-dimensional network in the primary plant cell wall. Current methods to assay XET activity use the XG polysaccharide as the donor substrate, and present limitations for kinetic and mechanistic studies of XET action due to the polymeric and polydisperse nature of the substrate. A novel activity assay based oil HPCE (high performance capillary electrophoresis), in con, junction with a defined low-molecular-mass XGO {XG oligosaccharicle; (XXXGXXXG, where G = Glc beta 1,4- and X = [Xyl alpha 1,6]Glc beta 1,4-)l as the glycosyl donor and a heptasaccharide derivatized with ANTS [8-aminonaphthalene-1,3,6-trisulphonic acid; (XXXG-ANTS)] as the acceptor substrate was developed and validated. The recombinant enzyme PttXET16A from Popidus tremula x tremuloides (hybrid aspen) was characterized using file donor/acceptor pair indicated above, for which preparative scale syntheses have been optimized. The low-molecular-mass donor underwent a single transglycosylation reaction to the acceptor substrate under initial-rate conditions. with a pH optimum at 5.0 and maximal activity between 30 and 40 degrees C. Kinetic data are best explained by a ping-pong bi-bi mechanism With Substrate inhibition by both donor and acceptor. This is the first assay for XETs using a donor Substrate other than polymeric XG, enabling quantitative kinetic analysis of different XGO donors for specificity, and subsite mapping studies of XET enzymes.
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