Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.772949
Title: The chemoenzymatic synthesis of the rare bacterial sugar pseudaminic acid, and its utilisation in the study of a potential pseudaminidase
Author: Chidwick, Harriet
ISNI:       0000 0004 7960 4033
Awarding Body: University of York
Current Institution: University of York
Date of Award: 2018
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Abstract:
Pseudaminic acid is a non-mammalian nonulosonic acid and a component in a number of bacterial surface structures, including Pseudomonas aeruginosa lipopolysaccharide and pili. It has been shown to play a role in virulence in pathogens such as influencing motility in Campylobacter jejuni whose flagellin is glycosylated with pseudaminic acid structures. Therefore pseudaminic acid processing enzymes have been identified as putative drug targets but are yet to be fully characterised. Although generally harmless to healthy individuals, Pseudomonas aeruginosa is the most prevalent lung disease in sufferers of cystic fibrosis and is implicated in the majority of cystic fibrosis deaths. Chronic infections are associated with progression into a mucoid phenotype whereby eradication of the pathogen is almost impossible. An enzyme associated with the mucoid phenotype (PA2794) has been putatively assigned as a pseudaminidase and this project aimed to unequivocally assign this enzyme using pseudaminic acid analogues. However strategies to synthesise pseudaminic acid are currently unsuitable for large scale (< 50 mg) production and hence chemical probes for the characterisation of pseudaminic acid processing enzymes are not currently available. It was attempted to attain a PA2794 crystal structure with pseudaminic acid in complex. However this was inconclusive in elucidating the PA2794 natural substrate and further investigations prevented due to the lack of availability of the putative ligand. Therefore efforts were turned towards the design of a strategy for the synthesis of pseudaminic acid on a large scale, to allow for the pseudaminic acid chemical probes to be developed. The proposed synthesis utilised the Campylobacter jejuni pseudaminic acid biosynthetic enzymes to convert UDP-GlcNAc into pseudaminic acid in one-pot. Expression was optimised for each enzyme and modified to ensure enzyme solubility. A coupled PseB, PseC activity assay was implemented to follow conversion to the unwanted PseB by-product, and conditions adjusted to perturb this reaction. Additionally co-factor substitutes were explored in order to make large scale synthesis economically viable. A route for the large scale enzymatic production of pseudaminic acid was optimised, allowing for an economically viable, efficient and high yielding synthesis.
Supervisor: Fascione, Martin ; Davies, Gideon Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.772949  DOI: Not available
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