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Differences in enzymic properties of five recombinant xyloglucan endotransglucosylase/hydrolase (XTH) proteins of Arabidopsis thaliana
KTH, School of Biotechnology (BIO), Glycoscience.
KTH, School of Biotechnology (BIO), Glycoscience.
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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.

Place, publisher, year, edition, pages
2011. Vol. 62, no 1, 261-271 p.
Keyword [en]
Arabidopsis thaliana, Brassicaceae, cell elongation, cell wall, heterologous protein production, xyloglucan endotransglucosylase, hydrolase (XTH)
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-28187DOI: 10.1093/jxb/erq263ISI: 000284951900022OAI: oai:DiVA.org:kth-28187DiVA: diva2:385079
Funder
Swedish Research Council
Note
QC 20110111Available from: 2011-01-11 Created: 2011-01-10 Last updated: 2017-12-11Bibliographically approved
In thesis
1. In vitro and in vivo approaches in the characterization of XTH gene products
Open this publication in new window or tab >>In vitro and in vivo approaches in the characterization of XTH gene products
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

ABSTRACT

The xyloglucan endo-transglycosylase/hydrolase (XTH) genes are found in all vascular and some nonvascular plants. The XTH genes encode proteins which comprise a subfamily of glycoside hydrolase (GH) family 16 in the Carbohydrate-Active enZYmes (CAZY) classification. The XTH gene products are believed to play intrinsic role in cell wall modification during growth and development throughout the lifetime of the plant. In the present investigation, biochemical and reverse genetic approaches were used to better understand the functions of individual members of the XTH gene family of two important plants: the model organism Arabidopsis thaliana and the grain crop barley (Hordeum vulgare). A phylogenetic tree of the xyloglucan-active enzymes of GH16 has previously been constructed, where enzymes with similar activities have been shown to cluster together. Several members of phylogenetic Group I/II and III-B, predicted to exhibit xyloglucan endo-transglycosylase activity (EC 2.4.1.207) and members of Group III-A, predicted to exhibit xyloglucan endo-hydrolase activity (EC 3.2.1.151), were included to analyze the functional diversity of XTH gene products. A heterologous expression system using the yeast Pichia pastoris was found to be effective for recombinant protein production with a success rate of ca. 50%. XTH gene products were obtained in soluble and active forms for subsequent biochemical characterization.

In order to be able to screen larger numbers of protein producing clones, a fast and easy method is required to identify clones expressing active protein in high enough amounts. Thus, a miniaturized XET/XEH assay for high-throughput analysis was developed, which was able to identify activities with good precision and with a reduced time and materials consumption and a reduced work load.

Enzyme kinetic analysis indicated that the XET or XEH activity of all XTH gene products characterized in the present study corresponded to predictions based on the previously revised phylogenetic clustering. To gain insight into the biological function of the predominant XEHs AtXTH31 and AtXTH32, which are highly expressed in rapidly developing tissues, a reverse genetic approach was employed using T-DNA insertion lines of the A. thaliana Columbia ecotype. Genotypic and phenotypic characterization, together with in situ assays of XET and XEH activities, in single- and double-knock-out mutants indicated that these Group III-A enzymes are active in expanding tissues of the A. thaliana roots and hypocotyl.  Although suppression of in muro XEH activity was clearly observed in the double-knock-out, no significant growth phenotype was observed, with the exception that radicle emergence appeared to be faster than in the wild type plants.

Keywords: Arabidopis thaliana, Hordeum vulgare, plant cell wall, xyloglucan, glycoside hydrolase family 16, xyloglucan endo-transglycosylase/hydrolase gene family, xyloglucan endo-transglycosylase, xyloglucan endo-hydrolase, heterologous protein expression, Pichia pastoris, T-DNA insertion, in situ XET/XEH assay, high-throughput screening

Place, publisher, year, edition, pages
Stockholm: KTH, 2011. x, 48 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2011: 1
Keyword
Arabiodopsis thaliana, Hordeum vulgare, plant cell wall, xyloglucan, glycoside hydrolase family 16, xyloglucan endo-transglycosylase/hydrolase gene family, xyloglucan endo-hydrolase, heterologous protein expression, Pichia pastoris, T-DNA insertion, in situ XET/XEH assay, high-througput screening
National Category
Botany Biochemistry and Molecular Biology Biochemistry and Molecular Biology
Research subject
SRA - Molecular Bioscience
Identifiers
urn:nbn:se:kth:diva-28222 (URN)978-91-7415-848-9 (ISBN)
Public defence
2011-02-02, FB54, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20110114Available from: 2011-01-14 Created: 2011-01-12 Last updated: 2011-01-14Bibliographically approved
2. Plant and microbial xyloglucanases: Function, Structure and Phylogeny
Open this publication in new window or tab >>Plant and microbial xyloglucanases: Function, Structure and Phylogeny
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
xyloglucan, xyloglucanases, XTH, XET, XEH, endoglucanases, EG16
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-31677 (URN)978-91-7415-932-5 (ISBN)
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

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