The University of Waikato
The biochemistry and phylogeny of CysE: An antimicrobial target
Abstract
dc:description.abstractNeisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted infection (STI), gonorrhoea. There are over 87 million gonorrhoea cases per year globally, making it the second most prevalent bacterial STI. The rapid emergence of antimicrobial-resistant strains has led N. gonorrhoeae to being labelled as a priority pathogen for the development of new antimicrobials by the World Health Organisation. One strategy for identifying new antimicrobials is through targeting pathways required for virulence and mitigation of oxidative stress, such as sulphur assimilation pathways. This thesis investigates the potential of targeting the de novo cysteine biosynthetic enzyme, serine acetyltransferase, from N. gonorrhoeae (NgCysE) for new antimicrobials. In this thesis, we present the kinetic and structural characterisation of NgCysE. NgCysE has serine acetyltransferase activity and is sensitive to feedback inhibition by L cysteine. Small-angle X-ray scattering demonstrates NgCysE is a single hexameric species in solution with subtle conformational changes upon binding of its inhibitor L-cysteine. Using X-ray crystallography, we present two NgCysE structures, with L-malate (2.01 Å) and substrate L-serine (2.8 Å), bound. NgCysE crystallises as a homohexamer with 3:2-fold symmetry with each monomer containing a N-terminal α-helical SATase and C-terminal left-handed β-helix domain. Additionally, we see extensive density for the C-terminal α-helical tails because of domain swapping, resulting in one of the most complete CysE crystal structures to date. The high resolution NgCysE structure was used for structure-based virtual inhibitor screening to identify potential inhibitors of NgCysE. Virtual screening produced 28 hit compounds that were tested in vitro, with the most potent inhibitor, compound 2, demonstrating inhibition in the low micromolar range. Analysis of compound 2 docking reveals interactions with both NgCysE substrate binding sites, serine and acetyl-CoA. This is the first reported inhibitor of NgCysE and provides a starting point for development of new antimicrobials for treating gonorrhoea infection. Lastly, we present the phylogenetic analysis of truncated and full-length CysE isoforms across the bacterial kingdom. We demonstrate that a truncated isoform has a discrete truncation of four α-helices from the N-terminal SATase domain and is the predominant isoform for Gram-positive bacteria. The functional implications of this truncation are explored using predictive modelling and demonstrate that CysE is likely to exist as a trimer rather than the hexamer seen for the full-length isoform. This highlights the diversity of CysE isoforms and paves the way for determining the evolutionary origin of these isoforms. Collectively, this thesis highlights the merit in the emerging field of CysE inhibition. Moreover, we provide the basis for the future development of not only NgCysE inhibitors, but also the development of CysE inhibitors for other bacterial pathogens.
Degree
thesis:*- Grantor dc:publisher.institution
- The University of Waikato
- Year dc:date.issued
- 2024
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
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- Oldham, Keely
- Advisors dc:contributor.advisor
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- Hicks, Joanna
- Arcus, Vickery
Subjects
dc:subject × 4Rights
dc:rights- Statement dc:rights
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- All items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.