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Title: Glycosyltransferases of Arabidopsis thaliana in biocatalysis
Author: Weis, Markus
ISNI:       0000 0004 2718 8576
Awarding Body: University of York
Current Institution: University of York
Date of Award: 2006
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Glycosyltransferases (GTs) constitute a large family of enzymes that are involved in the biosynthesis of a diversity of glycoconjugates. Family 1 GTs catalyse the transfer of sugars from activated sugar donors to a number of small lipophilic acceptor molecules. In the Arabidopsis genome, one hundred and seven GT-encoding genes from Family 1 have been identified by sequence analysis. The in vitro analysis of their gene products revealed that Arabidopsis thaliana (A. thaliana) GTs form glycosides of a number of plant secondary metabolites, phytohormones and of non-natural small molecules (xenobiotics) with high selectivity. When A. thaliana GTs were expressed in E. coli cells, their utility as whole-cell biocatalysts for the production of selective glycoconjugates has been demonstrated. This approach combines two advantages: first, the use of activated donor sugars for the reaction derived from the biosynthetic pool present in E. coli and second, recovery of the formed glycoside in the culture medium followed by classical purification methods. In this context, A. thaliana GTs may have the potential for development into biocatalysts for the synthesis of glycosides of pharmaceutical and nutraceutical importance. Therefore, a rapid screening method applicable for GTs active in whole-cell biocatalysis was developed such that one could scale-up rapidly from identification of a GT in a 96-well plate screen to use of the enzyme in a fermenter system for the synthesis of glycoconjugates (Chapter 3). The application of the whole-cell screen towards three aromatic substrates, trans-resveratrol, podophyllotoxin and daidzein, identified twenty-five and thirteen GTs with interesting selectivities that recognise trans-resveratrol and daidzein respectively (Chapter 4). Structural analyses of GTs revealed two structural superfamilies, named according to their fold GT-A or GT-B. Structures of five Family 1 GTs have been solved and shown to consist of the GT-B topology. A GT-B fold comprises two domains; the C-terminal domain responsible for donor sugar binding whereas the N-terminal binds the acceptor molecule. Since it is believed that within a family the same fold occurs, A. thaliana GTs should be a member of the GT-8 superfamily. Therefore, this study also investigated the role of either domain in sugar donor and acceptor recognition by exchanging the N-terminal domains between two similar A. thaliana GTs followed by a detailed kinetic analysis of the mutants (Chapter 5).
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available