| 1. |
|
|
| 2. |
- Baumann, Martin J., et al.
(författare)
-
Structural evidence for the evolution of xyloglucanase activity from xyloglucan endo-transglycosylases : Biological implications for cell wall metabolism
- 2007
-
Ingår i: The Plant Cell. - : Oxford University Press (OUP). - 1040-4651 .- 1532-298X. ; 19:6, s. 1947-1963
-
Tidskriftsartikel (refereegranskat)abstract
- High-resolution, three-dimensional structures of the archetypal glycoside hydrolase family 16 (GH16) endo-xyloglucanases Tm-NXG1 and Tm-NXG2 from nasturtium (Tropaeolum majus) have been solved by x-ray crystallography. Key structural features that modulate the relative rates of substrate hydrolysis to transglycosylation in the GH16 xyloglucan-active enzymes were identified by structure-function studies of the recombinantly expressed enzymes in comparison with data for the strict xyloglucan endo-transglycosylase Ptt-XET16-34 from hybrid aspen ( Populus tremula 3 Populus tremuloides). Production of the loop deletion variant Tm-NXG1-Delta YNIIG yielded an enzyme that was structurally similar to Ptt- XET16-34 and had a greatly increased transglycosylation: hydrolysis ratio. Comprehensive bioinformatic analyses of XTH gene products, together with detailed kinetic data, strongly suggest that xyloglucanase activity has evolved as a gain of function in an ancestral GH16 XET to meet specific biological requirements during seed germination, fruit ripening, and rapid wall expansion.
|
|
| 3. |
|
|
| 4. |
- Bollok, Monika, et al.
(författare)
-
Production of poplar xyloglucan endotransglycosylase using the methylotrophic yeast Pichia pastoris
- 2005
-
Ingår i: Applied Biochemistry and Biotechnology. - : Springer Science and Business Media LLC. - 0273-2289 .- 1559-0291. ; 126, s. 61-77
-
Tidskriftsartikel (refereegranskat)abstract
- The gene XET16A encoding the enzyme xyloglucan endotransglycosylase (XET) from hybrid aspen (Populus tremula x tremuloides Mich) was transformed into Pichia pastoris GS115 and the enzyme was secreted to the medium. The influence of process conditions on the XET production, activity, and proteolytic degradation were examined. Inactivation of XET occurred in the foam, but could be decreased significantly by using an efficient antifoam. Rich medium (yeast extract plus peptone) was needed for product accumulation, but not for growth. The proteolytic degradation of the enzyme in the medium was substantially decreased by also adding yeast extract and peptone to the glycerol medium before induction with methanol. Decreasing the fermentation pH from 5.0 to 4.0 further reduced the proteolysis. The specific activity was further improved by production at 15 degrees C instead of 22 degrees C. In this way a XET production of 54 mg/L active enzyme could be achieved in the process with a specific activity of 18 Unit/mg protein after a downstream process including centrifugation, micro- and ultrafiltration, and ion exchange chromatography.
|
|
| 5. |
- Derba-Maceluch, Marta, et al.
(författare)
-
Suppression of xylan endotransglycosylase PtxtXyn10A affects cellulose microfibril angle in secondary wall in aspen wood
- 2015
-
Ingår i: New Phytologist. - : Wiley. - 0028-646X .- 1469-8137. ; 205:2, s. 666-681
-
Tidskriftsartikel (refereegranskat)abstract
- Certain xylanases from family GH10 are highly expressed during secondary wall deposition, but their function is unknown. We carried out functional analyses of the secondary-wall specific PtxtXyn10A in hybrid aspen (Populus tremulaxtremuloides).PtxtXyn10A function was analysed by expression studies, overexpression in Arabidopsis protoplasts and by downregulation in aspen.PtxtXyn10A overexpression in Arabidopsis protoplasts resulted in increased xylan endotransglycosylation rather than hydrolysis. In aspen, the enzyme was found to be proteolytically processed to a 68kDa peptide and residing in cell walls. Its downregulation resulted in a corresponding decrease in xylan endotransglycosylase activity and no change in xylanase activity. This did not alter xylan molecular weight or its branching pattern but affected the cellulose-microfibril angle in wood fibres, increased primary growth (stem elongation, leaf formation and enlargement) and reduced the tendency to form tension wood. Transcriptomes of transgenic plants showed downregulation of tension wood related genes and changes in stress-responsive genes. The data indicate that PtxtXyn10A acts as a xylan endotransglycosylase and its main function is to release tensional stresses arising during secondary wall deposition. Furthermore, they suggest that regulation of stresses in secondary walls plays a vital role in plant development.
|
|
| 6. |
- Filonova, Lada, et al.
(författare)
-
Analysis of the Surfaces of Wood Tissues and Pulp Fibers Using Carbohydrate-Binding Modules Specific for Crystalline Cellulose and Mannan
- 2007
-
Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 8:1, s. 91-97
-
Tidskriftsartikel (refereegranskat)abstract
- Carbohydrate binding modules (CBMs) are noncatalytic substrate binding domains of many enzymes involved in carbohydrate metabolism. Here we used fluorescent labeled recombinant CBMs specific for crystalline cellulose (CBM1HjCel7A) and mannans (CBM27TmMan5 and CBM35CjMan5C) to analyze the complex surfaces of wood tissues and pulp fibers. The crystalline cellulose CBM1HjCel7A was found as a reliable marker of both bacterially produced and plant G-layer cellulose, and labeling of spruce pulp fibers with CBM1HjCel7A revealed a signal that increased with degree of fiber damage. The mannan-specific CBM27TmMan5 and CBM35CjMan5C CBMs were found to be more specific reagents than a monoclonal antibody specific for (1→4)-β-mannan/galacto-(1→4)-β-mannan for mapping carbohydrates on native substrates. We have developed a quantitative fluorometric method for analysis of crystalline cellulose accumulation on fiber surfaces and shown a quantitative difference in crystalline cellulose binding sites in differently processed pulp fibers. Our results indicated that CBMs provide useful, novel tools for monitoring changes in carbohydrate content of nonuniform substrate surfaces, for example, during wood or pulping processes and possibly fiber biosynthesis.
|
|
| 7. |
|
|
| 8. |
- Filonova, Lada, et al.
(författare)
-
Mapping of crystalline cellulose and mannan on the surfaces of wood tissues and pulp fibers using carbohydrate binding modules
- 2007
-
Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 8:1, s. 91-97
-
Tidskriftsartikel (refereegranskat)abstract
- Carbohydrate binding modules (CBMs) are noncatalytic substrate binding domains of many enzymes involved in carbohydrate metabolism. Here we used fluorescent labeled recombinant CBMs specific for crystalline cellulose (CBM1(HjCel7A)) and mannans (CBM27(TmMan5) and CBM35(CjMan5C)) to analyze the complex surfaces of wood tissues and pulp fibers. The crystalline cellulose CBM1(HjCel7A) was found as a reliable marker of both bacterially produced and plant G-layer cellulose, and labeling of spruce pulp fibers with CBM1(HjCel7A) revealed a signal that increased with degree of fiber damage. The mannan-specific CBM27(TmMan5) and CBM35(CjMan5C) CBMs were found to be more specific reagents than a monoclonal antibody specific for (1 -> 4)-beta-mannan/galacto-(1 -> 4)-beta-mannan for mapping carbohydrates on native substrates. We have developed a quantitative fluorometric method for analysis of crystalline cellulose accumulation on fiber surfaces and shown a quantitative difference in crystalline cellulose binding sites in differently processed pulp fibers. Our results indicated that CBMs provide useful, novel tools for monitoring changes in carbohydrate content of nonuniform substrate surfaces, for example, during wood or pulping processes and possibly fiber biosynthesis.
|
|
| 9. |
|
|
| 10. |
|
|
| 11. |
- Kallas, Åsa
(författare)
-
Heterologous expression and characterization of xyloglucan endotransglycosylases from Populus tremula x tremuloides
- 2004
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Xyloglucan endotransglycosylases (XETs) are enzymes involvedin the reorganization of the primary cell wall. This thesisdescribes several aspects of XET16A from hybrid aspen (Populustremula x tremuloides). The gene was isolated from the hybridaspen cDNA library and the protein was expressed heterologouslyin the methylotrophic yeast Pichia pastoris. The yield ofpurified, active PttXET16A from shake flask cultivations wasaround 10 - 20 mg/litre. Different characteristics, such asstability, temperature- and pH optimum were studied. We useddeglycosylated PttXET16A wild type enzyme as well as a mutatedenzyme to elucidate the role and the importance of theglycosylation of the protein. The three dimensional structureof PttXET16A was determined by X-ray crystallography. This wasthe first XET structure to be published and it contributed to abetter understanding of the mechanism and substrate specificityof the enzyme. Keywords:xyloglucan endotransglycosylase,transglycosylation, hybrid aspen, cell expansion, heterologousexpression, Pichia pastoris, N-glycosylation, X-raycrystallography
|
|
| 12. |
- Kallas, Åsa, 1974-
(författare)
-
Heterologous expression, characterization and applications of carbohydrate active enzymes and binding modules
- 2006
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Wood and wood products are of great economical and environmental importance, both in Sweden and globally. Biotechnology can be used both for achieving raw material of improved quality and for industrial processes such as biobleaching. Despite the enormous amount of carbon that is fixed as wood, the knowledge about the enzymes involved in the biosynthesis, re-organization and degradation of plant cell walls is relatively limited. In order to exploit enzymes more efficiently or to develop new biotechnological processes, it is crucial to gain a better understanding of the function and mechanism of the enzymes. This work has aimed to increase the knowledge about some of the enzymes putatively involved in the wood forming processes in Populus. Xyloglucan endotransglycosylases and a putative xylanase represent transglycosylating and hydrolytic enzymes, respectively. Carbohydrate binding modules represent non-catalytic modules, which bind to the substrate. Among 24 genes encoding for putative xyloglucan endotransglycosylases or xyloglucan endohydrolases that were identified in the Populus EST database, two were chosen for further studies (PttXTH16-34 and PttXTH16-35). The corresponding proteins, PttXET16-34 and PttXET16-35, were expressed in P. pastoris, purified and biochemically characterized. The importance of the N-glycans was investigated by comparing the recombinant wild-type proteins with their deglycosylated counterparts. In order to obtain the large amounts of PttXET16-34 that were needed for crystallization and development of biotechnological applications, the conditions for the large-scale production of PttXET16-34 in a fermenter were optimized. In microorganisms, endo-(1,4)-β-xylanases are important members of the xylan degrading machinery. These enzymes are also present in plants where they might fulfill a similar, but probably more restrictive function. One putative endo-(1,4)-β-xylanase, denoted PttXYN10A, was identified in the hybrid aspen EST library. Sequence analysis shows that this protein contains three putative carbohydrate-binding modules (CBM) from family 22 in addition to the catalytic module from GH10. Heterologous expression and reverse genetics were applied in order to elucidate the function of the catalytic module as well as the binding modules of PttXYN10A. Just as in microorganisms, some of the carbohydrate active enzymes from plants have one or more CBM attached to the catalytic module. So far, a very limited number of plant CBMs has been biochemically characterized. A detailed bio-informatic analysis of the CBM family 43 revealed interesting modularity patterns. In addition, one CBM43 (CBM43PttGH17_84) from a putative Populus b-(1,3)-glucanase was expressed in E. coli and shown to bind to laminarin (β-(1,3)-glucan), mixed-linked β-(1,3)(1,4)-glucans and crystalline cellulose. Due to their high specificity for different carbohydrates, CBMs can be used as probes for the analysis of plant materials. Generally, they are more specific than both staining techniques and carbohydrate-binding antibodies. We have used cellulose- and mannan binding modules from microorganisms as tools for the analysis of intact fibers as well as processed pulps.
|
|
| 13. |
|
|
| 14. |
|
|
| 15. |
- Kallas, Åsa M., et al.
(författare)
-
Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremula x tremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris
- 2005
-
Ingår i: Biochemical Journal. - 0264-6021. ; 390, s. 105-113
-
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.
|
|
| 16. |
- Mark, Pekka, et al.
(författare)
-
Analysis of nasturtium TmNXG1 complexes by crystallography and molecular dynamics provides detailed insight into substrate recognition by family GH16 xyloglucan endo-transglycosylases and endo-hydrolases
- 2009
-
Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 75:4, s. 820-836
-
Tidskriftsartikel (refereegranskat)abstract
- Reorganization and degradation of the wall crosslinking and seed storage polysaccharide xyloglucan by glycoside hydrolase family 16 (GH16) endo-transglycosylases and hydrolases are crucial to the growth of the majority of land plants, affecting processes as diverse as germination, morphogenesis, and fruit ripening. A high-resolution, three-dimensional structure of a nasturtium (Tropaeolum majus) endo-xyloglucanase loop mutant, TmNXG1-Delta YNIIG, with an ohgosaccharide product bound in the negative active-site subsites, has been solved by X-ray crystallography. Comparison of this novel complex to that of the strict xyloglucan endotransglycosylase PttXET16-34 from hybrid aspen (Populus tremula x tremuloides), previously solved with a xylogluco-oligosaccharide bound in the positive subsites, highlighted key protein structures that affect the disparate catalytic activities displayed by these closely related enzymes. Combination of these "partial" active-site complexes through molecular dynamics simulations in water allowed modeling of wild-type TmNXG1, TmNXG1-Delta YNIIG, and wild-type PttXET16-34 in complex with a xyloglucan octadecasaccharide spanning the entire catalytic cleft. A comprehensive analysis of these full-length complexes underscored the importance of various loops lining the active site. Subtle differences leading to a tighter hydrogen bonding pattern on the negative (glycosyl donor) binding subsites, together with loop flexibility on the positive (glycosyl acceptor) binding subsites appear to favor hydrolysis over transglycosylation in GH16 xyloglucan-active enzymes.
|
|
| 17. |
|
|
| 18. |
|
|
