Structural and functional characterisation of bacterial polysaccharide synthesis and transport in health and disease.

Ashworth, Gareth (2020) Structural and functional characterisation of bacterial polysaccharide synthesis and transport in health and disease. Doctoral thesis, University of East Anglia.

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The Pseudomonas aeruginosa pel operon is involved in pathogenic biofilm formation via synthesis of the Pel exopolysaccharide. Described here is the cloning and expression of the seven proteins of the Pel operon. A 2.6 Å resolution structure of an N-terminal 53 kDa fragment of PelA has been solved, revealing an α-1,4-N-acetylgalactosaminidase domain with an unusual (βα)7 TIM barrel fold and a second domain belonging to the glutamine amidotransferase-like superfamily which contains a degenerate active site and mediates oligomerisation of PelA into a trimeric channel.

The glycosyltransferase WaaB from Salmonella enterica serotype Typhmirium is involved in essential lipopolysaccharide synthesis. Key residues were identified from the previously-solved structure of UDP-bound WaaB (PDB: 5N80). These residues were mutated and investigated using a glycosyltransferase assay for activity and five were found to contribute significantly towards activity. The T273A mutant was found to enhance activity and was further investigated to determine that this does not increase substrate promiscuity. The structure of this mutant co-crystallised with UDP was determined at 2.5 Å, revealing that WaaB T273A adopts a tighter, closed conformation in comparison to the open conformation of the wild type protein, allowing the identification of F13 and S265 as potentially important to protein activity and providing evidence against membrane-associated activation of WaaB.

Lactobacillus reuteri forms symbiotic biofilms within the guts of host organisms. The glycosyltransferase GtfC from Lactobacillus reuteri strains 100-23 and 53608 share ~97% sequence homology but show specific activity towards UDP-glucose and UDP-N-acetylglucosamine, respectively. The crystal structure of GtfC100-23 was determined at 2.6 Å resolution in both the apo form and in complex with UDP. Key residues involved in catalysis, catalysis-related tetramerization and acceptor and donor substrate binding have been identified for functional analysis by collaborators. Candidate residues responsible for UDP-sugar specificity have been identified as L174, S238 and F240.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User: Chris White
Date Deposited: 03 Jun 2021 13:35
Last Modified: 31 Dec 2021 01:38


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