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- 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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| 3. |
- 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. ; 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 A (=0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 A contact between N(epsilon2) and O(epsilon1). The pH variation of k(cat)/K(m) for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wild-type 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 non-productive 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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| 4. |
- 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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| 7. |
- von Ossowski, I., et al.
(författare)
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Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Ce17A. A comparison with Phanerochaete chrysosporium Cel7D
- 2003
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Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 333:4, s. 817-829
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Tidskriftsartikel (refereegranskat)abstract
- The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations. The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K-M and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3) + Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.
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