Change search
ReferencesLink to record
Permanent link

Direct link
Plant and microbial xyloglucanases: Function, Structure and Phylogeny
KTH, School of Biotechnology (BIO), Glycoscience. (Carbohydrate Enzymology Research Group)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology , 2011. , 61 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2011:07
Keyword [en]
xyloglucan, xyloglucanases, XTH, XET, XEH, endoglucanases, EG16
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-31677ISBN: 978-91-7415-932-5OAI: oai:DiVA.org:kth-31677DiVA: diva2:405550
Public defence
2011-04-15, FR4, Albanova Universitetscentrum, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20110401Available from: 2011-04-01 Created: 2011-03-22 Last updated: 2011-11-03Bibliographically approved
List of papers
1. Structure and Activity of a Paenibacillus polymyxa Xyloglucanase from Glycoside Hydrolase Family 44
Open this publication in new window or tab >>Structure and Activity of a Paenibacillus polymyxa Xyloglucanase from Glycoside Hydrolase Family 44
Show others...
2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 39, 33890-33900 p.Article in journal (Refereed) Published
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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-31964 (URN)10.1074/jbc.M111.262345 (DOI)000295159200021 ()2-s2.0-80053190600 (ScopusID)
Note
QC 20111103 Available from: 2011-04-01 Created: 2011-04-01 Last updated: 2011-11-03Bibliographically approved
2. Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH12
Open this publication in new window or tab >>Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH12
Show others...
2007 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 26, 19177-19189 p.Article in journal (Refereed) Published
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.

Keyword
plant-cell wall, carotovora subsp carotovora, angstrom resolution, cellulose surfaces, crystal-structures, endoglucanase, oligosaccharides, insights, cloning, enzyme
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-16723 (URN)10.1074/jbc.M700224200 (DOI)000247475300059 ()2-s2.0-34547134972 (ScopusID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2011-04-01Bibliographically approved
3. Structural evidence for the evolution of xyloglucanase activity from xyloglucan endo-transglycosylases: Biological implications for cell wall metabolism
Open this publication in new window or tab >>Structural evidence for the evolution of xyloglucanase activity from xyloglucan endo-transglycosylases: Biological implications for cell wall metabolism
Show others...
2007 (English)In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 19, no 6, 1947-1963 p.Article in journal (Refereed) Published
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.

Keyword
tropaeolum-majus l, germinated nasturtium seeds, glycoside hydrolases, expression analysis, kappa-carrageenase, sequence alignment, crystal-structures, pichia-pastoris, hybrid aspen, endotransglycosylase
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-16832 (URN)10.1105/tpc.107.051391 (DOI)000248451900017 ()2-s2.0-34547657101 (ScopusID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2011-04-01Bibliographically approved
4. Group III-A XTH genes encode predominant xyloglucan endo hydrolase active in expanding tissues of Arabidopsis thaliana
Open this publication in new window or tab >>Group III-A XTH genes encode predominant xyloglucan endo hydrolase active in expanding tissues of Arabidopsis thaliana
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Genetics
Identifiers
urn:nbn:se:kth:diva-28352 (URN)
Note
QC 20110114Available from: 2011-01-14 Created: 2011-01-14 Last updated: 2011-04-01Bibliographically approved
5. Differences in enzymic properties of five recombinant xyloglucan endotransglucosylase/hydrolase (XTH) proteins of Arabidopsis thaliana
Open this publication in new window or tab >>Differences in enzymic properties of five recombinant xyloglucan endotransglucosylase/hydrolase (XTH) proteins of Arabidopsis thaliana
Show others...
2011 (English)In: Journal of Experimental Botany, ISSN 0022-0957, E-ISSN 1460-2431, Vol. 62, no 1, 261-271 p.Article in journal (Refereed) Published
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.

Keyword
Arabidopsis thaliana, Brassicaceae, cell elongation, cell wall, heterologous protein production, xyloglucan endotransglucosylase, hydrolase (XTH)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-28187 (URN)10.1093/jxb/erq263 (DOI)000284951900022 ()
Funder
Swedish Research Council
Note
QC 20110111Available from: 2011-01-11 Created: 2011-01-10 Last updated: 2011-04-01Bibliographically approved
6. An endo β‐1,4 glucanse, PtEG16‐1 from black cottonwood (Populustrichocarpa) represents an evolutionary link between bacterial lichenases and XTH geneproducts
Open this publication in new window or tab >>An endo β‐1,4 glucanse, PtEG16‐1 from black cottonwood (Populustrichocarpa) represents an evolutionary link between bacterial lichenases and XTH geneproducts
(English)Article in journal (Other academic) Submitted
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-31985 (URN)
Note
QS 20120326Available from: 2011-04-01 Created: 2011-04-01 Last updated: 2012-03-26Bibliographically approved

Open Access in DiVA

fulltext(6819 kB)1384 downloads
File information
File name FULLTEXT01.pdfFile size 6819 kBChecksum SHA-512
d364e21a41407bf4c3617e7ce922d43fb7a0b6c9f96425b26ad3c7d75941c0463bdbd5c3983406ea1e7ed78aba927cde5baea776152874be57e9e800c0ed1b7e
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Eklöf, Jens
By organisation
Glycoscience
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 1384 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Total: 1940 hits
ReferencesLink to record
Permanent link

Direct link