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A holistic approach to understanding CAZy families through reductionist methods
KTH, School of Biotechnology (BIO), Glycoscience.
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 [en]
Carbohydrate esterase family 8, XET, PME, YbhC
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-10183ISBN: 978-91-7415-269-2 (print)OAI: oai:DiVA.org:kth-10183DiVA: diva2:210065
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
List of papers
1. The crystal structure of the outer membrane lipoprotein YbhC from Escherichia coli sheds new light on the phylogeny of carbohydrate esterase family 8
Open this publication in new window or tab >>The crystal structure of the outer membrane lipoprotein YbhC from Escherichia coli sheds new light on the phylogeny of carbohydrate esterase family 8
2009 (English)In: Proteins: Structure, Function, and Genetics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 76, no 4, 1029-1036 p.Article in journal (Refereed) Published
Keyword
carbohydrate-active enzyme (CAZyme), CE8, pectin methylesterase (PME), palmitoyl coenzyme A, murein, Enterobacteriaceae, penicillin-binding protein-5, pectin methylesterase, maximum-likelihood, sequence, identification, specificity, models
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-18676 (URN)10.1002/prot.22453 (DOI)000268839400020 ()2-s2.0-68149162197 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2010-12-21Bibliographically approved
2. 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 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2011-04-01Bibliographically approved

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