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  • 1. Ariza, A.
    et al.
    Eklöf, Jens
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Spadiut, Oliver
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Offen, W.A.
    Roberts, S.M.
    Wilson, K.S.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Davies, G.J.
    Structure and Activity of a Paenibacillus polymyxa Xyloglucanase from Glycoside Hydrolase Family 442011Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, nr 39, s. 33890-33900Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The enzymatic degradation of plant polysaccharides is emerging as one of the key environmental goals of the early 21st century, impacting on many processes in the textile and detergent industries as well as biomass conversion to biofuels. One of the well known problems with the use of nonstarch (nonfood)-based substrates such as the plant cell wall is that the cellulose fibers are embedded in a network of diverse polysaccharides, including xyloglucan, that renders access difficult. There is therefore increasing interest in the "accessory enzymes," including xyloglucanases, that may aid biomass degradation through removal of "hemicellulose" polysaccharides. Here, we report the biochemical characterization of the endo-beta-1,4-(xylo)glucan hydrolase from Paenibacillus polymyxa with polymeric, oligomeric, and defined chromogenic aryl-oligosaccharide substrates. The enzyme displays an unusual specificity on defined xyloglucan oligosaccharides, cleaving the XXXG-XXXG repeat into XXX and GXXXG. Kinetic analysis on defined oligosaccharides and on aryl-glycosides suggests that both the -4 and +1 subsites show discrimination against xylose-appended glucosides. The three-dimensional structures of PpXG44 have been solved both in apo-form and as a series of ligand complexes that map the -3 to -1 and +1 to +5 subsites of the extended ligand binding cleft. Complex structures are consistent with partial intolerance of xylosides in the -4' subsites. The atypical specificity of PpXG44 may thus find use in industrial processes involving xyloglucan degradation, such as biomass conversion, or in the emerging exciting applications of defined xyloglucans in food, pharmaceuticals, and cellulose fiber modification.

  • 2.
    Baumann, Martin J.
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Eklöf, Jens M.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Michel, Gurvan
    Kallas, Åsa M.
    KTH, Skolan för bioteknologi (BIO).
    Teeri, Tuula T.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Czjzek, Mirjam
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Structural evidence for the evolution of xyloglucanase activity from xyloglucan endo-transglycosylases: Biological implications for cell wall metabolism2007Inngår i: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 19, nr 6, s. 1947-1963Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.
    Baumann, Martin J.
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Eklöf, Jens
    KTH, Skolan för bioteknologi (BIO).
    Michel, G.
    Kallas, Åsa
    KTH.
    Teeri, Tuula
    KTH, Skolan för bioteknologi (BIO).
    Czjzek, Mirjam
    KTH.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Structural analysis of nasturtium NXG reveals the evolution of GH16 xyloglucanase activity from XETs: biological implications for cell wall metabolismManuskript (Annet vitenskapelig)
  • 4.
    Eklof, Jens M.
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. Michael Smith Laboratories, Department of Chemistry, University of British Columbia, Canada.
    Ruda, Marcus C.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. Michael Smith Laboratories, Department of Chemistry, University of British Columbia, Canada.
    Distinguishing xyloglucanase activity in endo-β(1 → 4)glucanases2012Inngår i: Methods in Enzymology, ISSN 0076-6879, E-ISSN 1557-7988, Vol. 510, s. 97-120Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The ability of beta-glucanases to cleave xyloglucans, a family of highly decorated beta-glucans ubiquitous in plant biomass, has traditionally been overlooked in functional biochemical studies. An emerging body of data indicates, however, that a spectrum of xyloglucan specificity resides in diverse glycoside hydrolases from a range of carbohydrate-active enzyme families including classic "cellulase" families. This chapter outlines a series of enzyme kinetic and product analysis methods to establish degrees of xyloglucan specificity and modes of action of glycosidases emerging from enzyme discovery projects.

  • 5.
    Eklöf, Jens
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    A holistic approach to understanding CAZy families through reductionist methods2009Licentiatavhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

     

    In a time when the amount of biological data present in the public domain is becoming increasingly vast, the need for good classification systems has never been greater. In the field of glycoscience the necessity of a good classification for the enzymes involved in the biosynthesis, modification and degradation of polysaccharides is even more pronounced than in other fields. This is due to the complexity of the substrates, the polysaccharides, as the theoretical number of possible hexa-oligosaccharides from only hexoses exceeds 1012 isomers! 

    An initiative to classify enzymes acting on carbohydrates began around 1990 by the French scientist Bernard Henrissat. The resulting database, the Carbohydrate Active enzymes database (CAZy), classifies enzymes by sequence similarity into families allowing the inference of structure and catalytic mechanism. What CAZy does not provide however, are means to understand how members of a family are related, and in what way they differ from each other. The top-down approach used in this thesis, combining phylogenetic analysis of whole CAZy families, or sub-families, with structural determinations and detailed kinetic analysis allows for exactly that.  

    Finding determinants for transglycosylation versus hydrolysis within the xth gene product family of GH16 as well as restricting the hydrolytic enzymes to a well defined clade are integral parts of paper I. In paper II a new bacterial sub-clade within CE8 was discovered. The structural determination of theEscherichia coli outer membrane lipoprotein YbhC from from the new sub-clade explained the difference in specificity. The information provided in the two papers of this thesis gives a better understanding of the development of different specificities of diverse CAZY families as well as it aids in future gene product annotations. Furthermore this work has begun to fill the white spots uncovered in the phylogenetic trees.

     

     

  • 6.
    Eklöf, Jens
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Plant and microbial xyloglucanases: Function, Structure and Phylogeny2011Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    In this thesis, enzymes acting on the primary cell wall hemicellulose xyloglucan are studied.  Xyloglucans are ubiquitous in land plants which make them an important polysaccharide to utilise for microbes and a potentially interesting raw material for various industries.  The function of xyloglucans in plants is mainly to improve primary cell wall characteristics by coating and tethering cellulose microfibrils together.  Some plants also utilise xyloglucans as storage polysaccharides in their seeds.

    In microbes, a variety of different enzymes for degrading xyloglucans have been found.  In this thesis, the structure-function relationship of three different microbial endo-xyloglucanases from glycoside hydrolase families 5, 12 and 44 are probed and reveal details of the natural diversity found in xyloglucanases.  Hopefully, a better understanding of how xyloglucanases recognise and degrade their substrate can lead to improved saccharification processes of plant matter, finding uses in for example biofuel production.

    In plants, xyloglucans are modified in muro by the xyloglucan transglycosylase/hydrolase (XTH) gene products.  Interestingly, closely related XTH gene products catalyse either transglycosylation (XET activity) or hydrolysis (XEH activity) with dramatically different effects on xyloglucan and on cell wall characteristics.  The strict transglycosylases transfer xyloglucan segments between individual xyloglucan molecules while the hydrolases degrade xyloglucan into oligosaccharides.  Here, we describe and determine, a major determinant of transglycosylation versus hydrolysis in XTH gene products by solving and comparing the first 3D structure of an XEH, Tm-NXG1 and a XET, PttXET16-34.  The XEH activity was hypothesised, and later confirmed to be restricted to subset of the XTH gene products.  The in situ localisation of XEH activity in roots and hypocotyls of Arabidopsis was also visualised for the first time.  Furthermore, an evolutionary scheme for how XTH gene products developed from bacterial beta-1,3;1,4 glucanases was also presented based on the characterisation of a novel plant endo-glucanase, PtEG16-1. The EG16s are proposed to predate XTH gene products and are with activity on both xyloglucan and beta-1,3;1,4 glucans an “intermediate” in the evolution from beta-1,3;1,4 glucanases to XTH gene products.

  • 7.
    Eklöf, Jens
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    An endo β‐1,4 glucanse, PtEG16‐1 from black cottonwood (Populustrichocarpa) represents an evolutionary link between bacterial lichenases and XTH geneproductsArtikkel i tidsskrift (Annet vitenskapelig)
  • 8.
    Eklöf, Jens M.
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    The XTH Gene Family: An Update on Enzyme Structure, Function, and Phylogeny in Xyloglucan Remodeling2010Inngår i: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 153, nr 2, s. 456-466Artikkel i tidsskrift (Fagfellevurdert)
  • 9.
    Eklöf, Jens M.
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Shojania, S.
    Okon, M.
    McIntosh, L. P.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Structure-function analysis of a broad specificity Populus trichocarpa endo-β-glucanase reveals an evolutionary link between bacterial licheninases and plant XTH gene products2013Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, nr 22, s. 15786-15799Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The large xyloglucan endotransglycosylase/hydrolase (XTH) gene family continues to be the focus of much attention in studies of plant cell wall morphogenesis due to the unique catalytic functions of the enzymes it encodes. The XTH gene products compose a subfamily of glycoside hydrolase family 16 (GH16), which also comprises a broad range of microbial endoglucanases and endogalactanases, as well as yeast cell wall chitin/β-glucan transglycosylases. Previous whole-family phylogenetic analyses have suggested that the closest relatives to the XTH gene products are the bacterial licheninases (EC 3.2.1.73), which specifically hydrolyze linear mixed linkage β(1→3)/β(1→4)-glucans. In addition to their specificity for the highly branched xyloglucan polysaccharide, XTH gene products are distinguished from the licheninases and other GH16 enzyme subfamilies by significant active site loop alterations and a large C-terminal extension. Given these differences, the molecular evolution of the XTH gene products in GH16 has remained enigmatic. Here, we present the biochemical and structural analysis of a unique, mixed function endoglucanase from black cottonwood (Populus trichocarpa), which reveals a small, newly recognized subfamily of GH16 members intermediate between the bacterial licheninases and plant XTH gene products. We postulate that this clade comprises an important link in the evolution of the large plant XTH gene families from a putative microbial ancestor. As such, this analysis provides new insights into the diversification of GH16 and further unites the apparently disparate members of this important family of proteins. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

  • 10.
    Eklöf, Jens M.
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Tan, Tien-Chye
    KTH, Skolan för bioteknologi (BIO).
    Divne, Christina
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    The crystal structure of the outer membrane lipoprotein YbhC from Escherichia coli sheds new light on the phylogeny of carbohydrate esterase family 82009Inngår i: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 76, nr 4, s. 1029-1036Artikkel i tidsskrift (Fagfellevurdert)
  • 11. Gloster, Tracey M.
    et al.
    Ibatullin, Farid M.
    Macauley, Katherine
    Eklöf, Jens
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Roberts, Shirley
    Turkenburg, Johan P.
    Bjornvad, Mads E.
    Jorgensen, Per Lina
    Danielsen, Steffen
    Johansen, Katja S.
    Borchert, Torben V.
    Wilson, Keith S.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Davies, Gideon J.
    Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH122007Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, nr 26, s. 19177-19189Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed hemicellulose. One such hemicellulose is xyloglucan, which displays a beta-1,4-linked D-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligand free and xyloglucan- oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (beta/alpha)(8) and beta-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the beta-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzymeis similarly competent in the degradation of unbranched glucans. In the case of the (beta/alpha)(8) GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered.

  • 12.
    Kaewthai, Nomchit
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Gendre, Delphine
    Eklöf, Jens M.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Ibatullin, Farid M.
    Ezcurra, Ines
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Bhalerao, Rishikesh P.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Group III-A XTH genes encode predominant xyloglucan endo hydrolase active in expanding tissues of Arabidopsis thalianaManuskript (preprint) (Annet vitenskapelig)
  • 13.
    Kaewthai, Nomchit
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Gendre, Delphine
    Eklöf, Jens M.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Ibatullin, Farid M.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Ezcurra, Ines
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Bhalerao, Rishikesh P
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Group III-A XTH Genes of Arabidopsis Encode Predominant Xyloglucan Endohydrolases That Are Dispensable for Normal Growth2013Inngår i: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 161, nr 1, s. 440-454Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The molecular basis of primary wall extension endures as one of the central enigmas in plant cell morphogenesis. Classical cell wall models suggest that xyloglucan endo-transglycosylase activity is the primary catalyst (together with expansins) of controlled cell wall loosening through the transient cleavage and religation of xyloglucan-cellulose cross links. The genome of Arabidopsis (Arabidopsis thaliana) contains 33 phylogenetically diverse XYLOGLUCAN ENDO-TRANSGLYCOSYLASE/HYDROLASE (XTH) gene products, two of which were predicted to be predominant xyloglucan endohydrolases due to clustering into group III-A. Enzyme kinetic analysis of recombinant AtXTH31 confirmed this prediction and indicated that this enzyme had similar catalytic properties to the nasturtium (Tropaeolum majus) xyloglucanase1 responsible for storage xyloglucan hydrolysis during germination. Global analysis of Genevestigator data indicated that AtXTH31 and the paralogous AtXTH32 were abundantly expressed in expanding tissues. Microscopy analysis, utilizing the resorufin beta-glycoside of the xyloglucan oligosaccharide XXXG as an in situ probe, indicated significant xyloglucan endohydrolase activity in specific regions of both roots and hypocotyls, in good correlation with transcriptomic data. Moreover, this hydrolytic activity was essentially completely eliminated in AtXTH31/AtXTH32 double knockout lines. However, single and double knockout lines, as well as individual overexpressing lines, of AtXTH31 and AtXTH32 did not demonstrate significant growth or developmental phenotypes. These results suggest that although xyloglucan polysaccharide hydrolysis occurs in parallel with primary wall expansion, morphological effects are subtle or may be compensated by other mechanisms. We hypothesize that there is likely to be an interplay between these xyloglucan endohydrolases and recently discovered apoplastic exo-glycosidases in the hydrolytic modification of matrix xyloglucans.

  • 14. Maris, An
    et al.
    Kaewthai, Nomchit
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Eklöf, Jens
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Miller, Janice G.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Fry, Stephen C.
    Verbelen, Jean-Pierre
    Vissenberg, Kris
    Differences in enzymic properties of five recombinant xyloglucan endotransglucosylase/hydrolase (XTH) proteins of Arabidopsis thaliana2011Inngår i: Journal of Experimental Botany, ISSN 0022-0957, E-ISSN 1460-2431, Vol. 62, nr 1, s. 261-271Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Xyloglucan endotransglucosylase/hydrolases (XTHs) are cell wall enzymes that are able to graft xyloglucan chains to oligosaccharides or to other available xyloglucan chains and/or to hydrolyse xyloglucan chains. As they are involved in the modification of the load-bearing cell-wall components, they are believed to be very important in the regulation of growth and development. Given the large number (33) of XTH genes in Arabidopsis and the overlapping expression patterns, specific enzymic properties may be expected. Five predominantly root-expressed Arabidopsis thaliana XTHs belonging to subgroup I/II were analysed here. These represent two sets of closely related genes: AtXTH12 and 13 on the one hand (trichoblast-enriched) and AtXTH17, 18, and 19 on the other (expressed in nearly all cell types in the root). They were all recombinantly produced in the yeast Pichia pastoris and partially purified by ammonium sulphate precipitation before they were subsequently all subjected to a series of identical in vitro tests. The kinetic properties of purified AtXTH13 were investigated in greater detail to rule out interference with the assays by contaminating yeast proteins. All five proteins were found to exhibit only the endotransglucosylase (XET; EC 2.4.1.207) activity towards xyloglucan and non-detectable endohydrolytic (XEH; EC 3.2.1.151) activity. Their endotransglucosylase activity was preferentially directed towards xyloglucan and, in some cases, water-soluble cellulose acetate, rather than to mixed-linkage beta-glucan. Isoforms differed in optimum pH (5.0-7.5), in temperature dependence and in acceptor substrate preferences.

  • 15.
    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-hydrolases2009Inngår i: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 75, nr 4, s. 820-836Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 16.
    Nordgren, Niklas
    et al.
    KTH, Skolan för kemivetenskap (CHE), Kemi, Yt- och korrosionsvetenskap.
    Eklöf, Jens
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Zhou, Qi
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Brumer III, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Rutland, Mark
    KTH, Skolan för kemivetenskap (CHE), Kemi, Yt- och korrosionsvetenskap.
    Top-Down Grafting of Xyloglucan to Gold Monitored by QCM-D and AFM: Enzymatic Activity and Interactions with Cellulose2008Inngår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 9, nr 3, s. 942-948Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    This study focuses on the manufacture and characterisation of model surfaces consisting of end grafted xyloglucan (XG), a naturally occurring polysaccharide, onto a gold substrate.  The now well-established XET-technology was utilised for enzymatic incorporation of a thiol moiety at one end of the xyloglucan backbone.  This functionalised macromolecule was subsequently top-down grafted to gold, forming a thiol-bonded xyloglucan brush-like layer. The grafting was monitored in-situ with QCM-D and a significant difference in the adsorbed/grafted amount between unmodified xyloglucan and the thiol-functionalised polymer was observed.  The grafted surface was demonstrated to be accessible to enzyme digestion using the plant endo-xyloglucanase TmNXG1.  The nanotribological properties towards cellulose of the untreated crystal, brush modified surface and enzyme exposed surfaces were compared with a view to understanding the role of xyloglucan in friction reduction.  Friction coefficients obtained by the AFM colloidal probe technique using a cellulose functionalised probe on the xyloglucan brush showed an increase of a factor of two after the enzyme digestion and this result is interpreted in terms of surface roughness.  Finally, the brush is shown to exhibit binding to cellulose despite its highly oriented nature.

  • 17. Stepper, Judith
    et al.
    Dabin, Jerome
    Eklöf, Jens M.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Thongpoo, Preeyanuch
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Kongsaeree, Prachumporn
    Taylor, Edward J.
    Turkenburg, Johan P.
    Brumer, Harry
    University of British Columbia, Canada.
    Davies, Gideon J.
    Structure and activity of the Streptococcus pyogenes family GH1 6-phospho-beta-glucosidase SPy15992013Inngår i: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 69, s. 16-23Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The group A streptococcus Streptococcus pyogenes is the causative agent of a wide spectrum of invasive infections, including necrotizing fasciitis, scarlet fever and toxic shock syndrome. In the context of its carbohydrate chemistry, it is interesting that S. pyogenes (in this work strain M1 GAS SF370) displays a spectrum of oligosaccharide-processing enzymes that are located in close proximity on the genome but that the in vivo function of these proteins remains unknown. These proteins include different sugar transporters (SPy1593 and SPy1595), both GH125 alpha-1,6- and GH38 alpha-1,3-mannosidases (SPy1603 and SPy1604), a GH84 beta-hexosaminidase (SPy1600) and a putative GH2 beta-galactosidase (SPy1586), as well as SPy1599, a family GH1 'putative beta-glucosidase'. Here, the solution of the three-dimensional structure of SPy1599 in a number of crystal forms complicated by unusual crystallographic twinning is reported. The structure is a classical (beta/alpha)(g)-barrel, consistent with CAZy family GH1 and other members of the GH-A clan. SPy1599 has been annotated in sequence depositions as a beta-glucosidase (EC 3.2.1.21), but no such activity could be found; instead, three-dimensional structural overlaps with other enzymes of known function suggested that SPy1599 contains a phosphate-binding pocket in the active site and has possible 6-phospho-beta-glycosidase activity. Subsequent kinetic analysis indeed showed that SPy1599 has 6-phospho-beta-glucosidase (EC 3.2.1.86) activity. These data suggest that SPy1599 is involved in the intracellular degradation of 6-phosphoglycosides, which are likely to originate from import through one of the organism's many phosphoenolpyruvate phosphotransfer systems (PEP-PTSs).

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