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Title: Accessing pseudaminic acid (Pse5Ac7Ac) containing glycosides through the characterisation of Pse5Ac7Ac processing enzymes
Author: Flack, Emily K. P.
ISNI:       0000 0004 8499 1138
Awarding Body: University of York
Current Institution: University of York
Date of Award: 2019
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Cell-surface carbohydrate pseudaminic acid (Pse5Ac7Ac) is known to contribute to the virulence of several multi-drug resistant bacterial pathogens. Pse5Ac7Ac and its derivatives are not commercially available in appreciable quantities and chemical synthesis of these molecules has proved to be challenging. Access to Pse5Ac7Ac and activated CMP-Pse5Ac7Ac has been a hindrance in studies into the biological significance of Pse5Ac7Ac, including Pse5Ac7Ac-processing enzymes, which may be novel therapeutic targets. This project aimed to characterise enzymes which process pseudaminic acid and to chemoenzymatically synthesise glycosides which contain pseudaminic acid. Firstly, nucleotide-activated pseudaminic acid (CMP-Pse5Ac7Ac) was produced via a chemoenzymatic synthesis route. Six recombinant biosynthetic enzymes were purified for use in this reaction. With CMP-Pse5Ac7Ac in-hand, a library of bacterial glcosyltransferases were assayed for activity with CMP-Pse5Ac7Ac as donor. Success from this initial screen led to the synthesis of glycosides containing β-linked Pse5Ac7Ac, mediated by promiscuous glycosyltransferases. Finally, a putative pseudaminyltransferase was recombinantly produced, through the construction of a fusion protein. Activity studies revealed that the enzyme was able to utilise CMP-Pse5Ac7Ac and the product of the reaction was analysed, to confirm that the enzyme functions as a retaining pseudaminyltransferase. To our knowledge the work presented herein details the first examples of chemoenzymatic synthesis of glycosides containing Pse5Ac7Ac and the first in vivo study of a pseudaminyltransferases to provide unequivocal functional characterisation of this novel class of enzyme.
Supervisor: Fascione, Martin A. ; Thomas, Gavin H. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available