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  • 1.
    Gullfot, Fredrika
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för bioteknologi (BIO), Centra, Strategiskt Centrum för Biomimetiska Material, BioMime.
    On the engineering of proteins: methods and applications for carbohydrate-active enzymes2010Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    This thesis presents the application of different protein engineering methods on enzymes and non-catalytic proteins that act upon xyloglucans. Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glucans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glucans such as cellulose.

    One important group of xyloglucan-active enzymes is encoded by the GH16 XTH gene family in plants, including xyloglucan endo-transglycosylases (XET) and xyloglucan endo-hydrolases (XEH). The molecular determinants behind the different catalytic routes of these homologous enzymes are still not fully understood. By combining structural data and molecular dynamics (MD) simulations, interesting facts were revealed about enzyme-substrate interaction. Furthermore, a pilot study was performed using structure-guided recombination to generate a restricted library of XET/XEH chimeras.

    Glycosynthases are hydrolytically inactive mutant glycoside hydrolases (GH) that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Different enzymes with xyloglucan hydrolase activity were engineered into glycosynthases, and characterised as tools for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.

    Carbohydrate-binding modules (CBM) are non-catalytic protein domains that bind to polysaccharidic substrates. An important technical application involves their use as molecular probes to detect and localise specific carbohydrates in vivo. The three-dimensional structure of an evolved xyloglucan binding module (XGBM) was solved by X-ray diffraction. Affinity-guided directed evolution of this first generation XGBM resulted in highly specific probes that were used to localise non-fucosylated xyloglucans in plant tissue sections.

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    On the engineering of proteins
  • 2.
    Gullfot, Fredrika
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Synthesis of xyloglucan oligo- and polysaccharides with glycosynthase technology2009Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glycans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glycans such as cellulose.

    Xyloglucan is widely used in bulk quantities in the food, textile and paper making industries. With an increasing interest in technically more advanced applications of xyloglucan, such as novel biocomposites, there is a need to understand and control the properties and interactions of xyloglucan with other compounds, to decipher the relationship between xyloglucan structure and function, and in particular the effect of different branching patterns. However, due to the structural heterogeneity of the polysaccharide as obtained from natural sources, relevant studies have not been possible to perform in practise. This fact has stimulated an interest in synthetic methods to obtain xyloglucan mimics and analogs with well-defined structure and decoration patterns.

    Glycosynthases are hydrolytically inactive mutant glycosidases that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Since its first conception in 1998, the technology is emerging as a useful tool in the synthesis of large, complex polysaccharides. This thesis presents the generation and characterisation of glycosynthases based on xyloglucanase scaffolds for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.

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    FULLTEXT01
  • 3.
    Gullfot, Fredrika
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Ibatullin, Farid
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Sundqvist, Gustav
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Davies, Gideon
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Functional Characterization of Xyloglucan Glycosynthases from GH7, GH12, and GH16 Scaffolds2009Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 10, nr 7, s. 1782-1788Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Glycosynthases, hydrolytically inactive mutant glycosidases that catalyze glycosylation reactions using glycosyl fluoride donor substrates, are emerging as useful tools for the synthesis of large, complex polysaccharides [Faijes, M.; Planas, A. Carbohydr. Res. 2007, 342, 1581-1594]. Guided by wild-type xyloglucanase activity, we have produced and characterized new glycosynthases for the synthesis of xyloglucan oligo- and polysaccharides, based on family GH7, GH12, and GH16 scaffolds. The Humicola insolens GH7 glycosynthase, HiCel7B E197S, is capable of synthesizing nongalactosylated, XXXG-based homoxyloglucan up to Mw 60000 [G = Glcβ(1→4); X = Xylα(1→6)Glcβ(1→4); L = Galβ(1→2)Xylα(1→6)Glcβ(1→4)], which is among the largest products so far obtained with glycosynthase technology. Novel glycosynthases based on the GH16 xyloglucan hydrolase from Tropaeolum majus (nasturtium), TmNXG1, are capable of synthesizing XLLG-based xyloglucan oligosaccharides at rates feasible for preparative synthesis, thus providing an essential expansion of product range. Finally, a new glycosynthase based on the recently characterized GH12 xyloglucanase from Bacillus licheniformis, BlXG12 E155A, can perform the condensation of xyloglucosyl fluorides, albeit at poor rates. Altogether, the high catalytic efficiency demonstrated by HiCel7B E197S and the extended product range provided by TmNXG1 E94A are key achievements toward a robust and versatile method for the preparative synthesis of homogeneous xyloglucans with regular substitution patterns not available in nature. Such compounds enable in vitro experimental studies regarding the role of particular structural elements for xyloglucan properties and its interaction with cellulose.

  • 4.
    Gullfot, Fredrika
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Tan, Tien-Chye
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    von Schantz, Laura
    Karlsson, Eva Nordberg
    Ohlin, Mats
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Divne, Christina
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    The crystal structure of XG-34, an evolved xyloglucan-specific carbohydrate-binding module2010Ingår i: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 78, nr 3, s. 785-789Artikel i tidskrift (Refereegranskat)
  • 5.
    Gullfot, Fredrika
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för bioteknologi (BIO), Centra, Strategiskt Centrum för Biomimetiska Material, BioMime.
    Teeri, Tuula
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Design of GH16 XET/XEH chimeric enzymes with SCHEMA: ManuscriptManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    This manuscript contains experimental data obtained during a pilot study for the application of the SCHEMA method for structure-guided recombination on PttXET16-34 and TmNXG1, a model system for the evolution of different catalytic routes of GH16 XETs and XEHs.

    A restricted library of PttXET16-34/TmNXG1 chimeras with high diversity and low calculated SCHEMA disruption was generated based on crossover points identified by the RASPP algorithm. Analysis of the library revealed a bias among certain regions to remain intact and recalcitrant to recombination, in particular the upper and lower β-sheet structures forming the part of the protein that binds the donor substrate. In contrast, sequence diversity was preferentially introduced at the N-terminus, the major part of the acceptor side of the protein, and most of the C-terminal extension characteristic to XET/XEH in the GH16 family. Finally, in order to test the predictive capacity of SCHEMA, six chimeras with low calculated disruption were chosen for subsequent cloning and expression in Pichia pastoris.

  • 6.
    Mark, Pekka
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Baumann, Martin J.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Eklöf, Jens M.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Gullfot, Fredrika
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Michel, Gurvan
    Kallas, Åsa M.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Teeri, Tuula T.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Czjzek, Mirjam
    Analysis of nasturtium TmNXG1 complexes by crystallography and molecular dynamics provides detailed insight into substrate recognition by family GH16 xyloglucan endo-transglycosylases and endo-hydrolases2009Ingår i: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 75, nr 4, s. 820-836Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 7.
    Piens, Kathleen
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Henriksson, Maria
    KTH, Skolan för bioteknologi (BIO).
    Gullfot, Fredrika
    Lopez, Marie
    Fauré, Régis
    Ibatullin, Farid M.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Teeri, Tuula T.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Driguez, Hugues
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Glycosynthase activity of hybrid aspen xyloglucan endo-transglycosylase PttXET16-34 nucleophile mutants2007Ingår i: Organic and Biomolecular Chemistry, ISSN 1477-0520, Vol. 5, nr 24Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Glycosynthases are active-site mutants of glycoside hydrolases that catalyse glycosyl transfer using suitable activated donor substrates without competing product hydrolysis ( S. M. Hancock, M. D. Vaughan and S. G. Withers, Curr. Opin. Chem. Biol., 2006, 10, 509-519). Site-directed mutagenesis of the catalytic nucleophile, Glu-85, of a Populus tremula x tremuloides xyloglucan endo-transglycosylase (PttXET16-34, EC 2.4.1.207) into alanine, glycine, and serine yielded enzymes with glycosynthase activity. Product analysis indicated that PttXET16-34 E85A in particular was able to catalyse regio- and stereospecific homo- and hetero- condensations of alpha-xylogluco-oligosaccharyl fluoride donors XXXG alpha F andXLLG alpha F to produce xyloglucans with regular sidechain substitution patterns. This substrate promiscuity contrasts that of the Humicola insolens Ce17B E197A glycosynthase, which was not able to polymerise the di-galactosylated substrate XLLG alpha F. The production of the PttXET16-34 E85A xyloglucosynthase thus expands the repertoire of glycosynthases to include those capable of synthesising structurally homogenenous xyloglucans

  • 8.
    Spadiut, Oliver
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Ibatullin, Farid M.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Peart, Jonelle
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Gullfot, Fredrika
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Martinez-Fleites, Carlos
    Ruda, Marcus
    Xu, Chunlin
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Sundqvist, Gustav
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Davies, Gideon J.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Building Custom Polysaccharides in Vitro with an Efficient, Broad-Specificity Xyloglucan Glycosynthase and a Fucosyltransferase2011Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 133, nr 28, s. 10892-10900Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The current drive for applications of biomass-derived compounds, for energy and advanced materials, has led to a resurgence of interest in the manipulation of plant polymers. The xyloglucans, a family of structurally complex plant polysaccharides, have attracted significant interest due to their intrinsic high affinity for cellulose, both in muro and in technical applications. Moreover, current cell wall models are limited by the lack of detailed structure-property relationships of xyloglucans, due to a lack of molecules with well-defined branching patterns. Here, we have developed a new, broad-specificity "xyloglucan glycosynthase", selected from active-site mutants of a bacterial endoxyloglucanase, which catalyzed the synthesis of high molar mass polysaccharides, with complex side-chain structures, from suitable glycosyl fluoride donor substrates. The product range was further extended by combination with an Arabidopsis thaliana alpha(1 -> 2)-fucosyltransferase to achieve the in vitro synthesis of fucosylated xyloglucans typical of dicot primary cell walls. These enzymes thus comprise a toolkit for the controlled enzymatic synthesis of xyloglucans that are otherwise impossible to obtain from native sources. Moreover, this study demonstrates the validity of a chemo-enzymatic approach to polysaccharide synthesis, in which the simplicity and economy of glycosynthase technology is harnessed together with the exquisite specificity of glycosyltransferases to control molecular complexity.

  • 9. von Schantz, Laura
    et al.
    Gullfot, Fredrika
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Scheer, Sebastian
    Filonova, Lada
    Gunnarsson, Lavinia Cicortas
    Flint, James E.
    Daniel, Geoffrey
    Nordberg-Karlsson, Eva
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Ohlin, Mats
    Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules2009Ingår i: BMC Biotechnology, ISSN 1472-6750, E-ISSN 1472-6750, Vol. 9Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Molecular evolution of carbohydrate binding modules (CBM) is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology. Results: Two modified proteins with greatly improved affinity for xyloglucan, a key polysaccharide abundant in the plant kingdom crucial for providing plant support, were generated. Both improved modules differ from other existing xyloglucan probes by binding to galactose-decorated subunits of xyloglucan. The usefulness of the evolved binders was verified by staining of plant sections, where they performed better than the xyloglucan-binding module from which they had been derived. They discriminated non-fucosylated from fucosylated xyloglucan as shown by their ability to stain only the endosperm, rich in non-fucosylated xyloglucan, but not the integument rich in fucosylated xyloglucan, on tamarind seed sections. Conclusion: We conclude that affinity maturation of CBM selected from molecular libraries based on the CBM4-2 scaffold is possible and has the potential to generate new analytical tools for detection of plant carbohydrates.

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