Glycosyltransferases (GTs) are a large enzyme family that are involved in the biosynthesis of complex carbohydrates and glycoconjugates. These glycosylation reactions are essential to many fundamental biological processes, including cellular adhesion, cell signalling and bacterial cell wall biosynthesis. The aim of this project was the development of operationally simple assays for GTs and their application for the identification and characterisation of novel GT inhibitors. The main focus was on the bacterial GT, α1,4-galactosyltransferase C (LgtC) which transfers galactose from uridine-diphosphate-galactose to the terminal lactose on the cell wall lipooligopolysacharide of Neisseria meningitidis. A fluorescence-based ligand displacement assay (LDA) was optimised and validated for compound library screening. Additionally, the applicability of the LDA was investigated with three other GTs: bovine β1,4-galactosyltransferase, TcdB from Clostridium difficile and NGT from Actinobacillus pleuropneumoniae. The validated LDA was used for screening two compound libraries (c. 400 compounds in total) against LgtC for the first time. Positive hits were identified from both screening campaigns and selected compounds from two structural classes were characterised. One of these inhibitor classes was identified as molecular aggregators, which inhibit LgtC non-specifically. Results from the enzymological characterisation of the second class of hit compounds suggest that these inhibitors were mixed-type inhibitors and inhibit LgtC in micromolar range with considerable potential for further development. Development of a novel biochemical assay was attempted, in order to characterise potential inhibitors from the compound screening. This assay was based on an unnatural fluorescent acceptor and coupling of the GT reaction to a glycosidase reaction. During the development of this novel biochemical assay, unexpected enzymological features of LgtC were discovered. LC- MS/MS was used to investigate unknown products originating from the LgtC-catalysed reaction. The results suggest LgtC transfers more than one sugar moiety to the unnatural acceptor. Additionally two existing biochemical assays: HPLC-based assay and phosphatase coupled assay were successfully optimised and validated for determining the kinetic parameters of the LgtC catalysed reaction.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:628481 |
| Date | January 2014 |
| Creators | Goos, Niina |
| Publisher | King's College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://kclpure.kcl.ac.uk/portal/en/theses/assays-for-lycosyltransferases(38b943a8-4f87-4f82-ba5e-923bc550fcb6).html |
Page generated in 0.0019 seconds