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Structural evidence for the evolution of xyloglucanase activity from xyloglucan endo-transglycosylases: Biological implications for cell wall metabolism
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO), Glycoscience.
KTH, School of Biotechnology (BIO).
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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.

Place, publisher, year, edition, pages
2007. Vol. 19, no 6, 1947-1963 p.
Keyword [en]
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: urn:nbn:se:kth:diva-16832DOI: 10.1105/tpc.107.051391ISI: 000248451900017Scopus ID: 2-s2.0-34547657101OAI: oai:DiVA.org:kth-16832DiVA: diva2:334875
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
In thesis
1. A holistic approach to understanding CAZy families through reductionist methods
Open this publication in new window or tab >>A holistic approach to understanding CAZy families through reductionist methods
2009 (English)Licentiate thesis, comprehensive summary (Other academic)
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.

 

 

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. viii, 55 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:5
Keyword
Carbohydrate esterase family 8, XET, PME, YbhC
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-10183 (URN)978-91-7415-269-2 (ISBN)
Presentation
2009-04-29, FA31, Albanova University Center, Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2009-05-13 Created: 2009-03-30 Last updated: 2010-10-27Bibliographically 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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Baumann, Martin J.Eklöf, Jens M.Kallas, Åsa M.Teeri, Tuula T.Brumer, Harry
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