title: The biochemistry and phylogeny of CysE: An antimicrobial target

authors: Oldham, Keely

subjects: CysE

abstract: Neisseria 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.

text: The biochemistry and phylogeny of CysE: An antimicrobial target Neisseria 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.

year: 2024

title: Evaluating research - Peer review team assessment and journal-based bibliographic measures: New Zealand PBRF research output scores in 2006

authors: Anderson, David L.

subjects: Bibliometrics

abstract: This paper concerns the relationship between the assessment of the research of individual academics by peer or expert review teams with a variety of bibliometric schemes based on journal quality weights. Specifically, for a common group of economists from New Zealand departments of economics the relationship between Performance-Based Research Fund (PBRF) Research Output measures for those submitting new research portfolios in 2006 are compared with evaluations of journal based research over the 2000-2005 assessment period. This comparison identifies the journal weighting schemes that appear most similar to PBRF peer evaluations. The paper provides an indication of the ‘power or aggressiveness’ of PBRF evaluations in terms of the weighting given to quality. The implied views of PBRF peer review teams are also useful in assessing common assumptions made in evaluating journal based research.

text: Evaluating research - Peer review team assessment and journal-based bibliographic measures: New Zealand PBRF research output scores in 2006 This paper concerns the relationship between the assessment of the research of individual academics by peer or expert review teams with a variety of bibliometric schemes based on journal quality weights. Specifically, for a common group of economists from New Zealand departments of economics the relationship between Performance-Based Research Fund (PBRF) Research Output measures for those submitting new research portfolios in 2006 are compared with evaluations of journal based research over the 2000-2005 assessment period. This comparison identifies the journal weighting schemes that appear most similar to PBRF peer evaluations. The paper provides an indication of the ‘power or aggressiveness’ of PBRF evaluations in terms of the weighting given to quality. The implied views of PBRF peer review teams are also useful in assessing common assumptions made in evaluating journal based research.

year: 2012-04

title: Exploring te ao Māori: The role of museums

authors: Clarkin-Phillips, Jeanette

subjects: museum

abstract: Museums offer many opportunities for developing knowledge of the bicultural heritage of Aotearoa New Zealand. This article reports on research involving a kindergarten located in a national museum. It discusses children’s growing understanding of te ao Māori (the world of Māori) through their regular visits to the collections and exhibits in the museum and suggests teachers in early childhood centres may find connecting with their local museum a valuable resource for enhancing bicultural practice.

text: Exploring te ao Māori: The role of museums Museums offer many opportunities for developing knowledge of the bicultural heritage of Aotearoa New Zealand. This article reports on research involving a kindergarten located in a national museum. It discusses children’s growing understanding of te ao Māori (the world of Māori) through their regular visits to the collections and exhibits in the museum and suggests teachers in early childhood centres may find connecting with their local museum a valuable resource for enhancing bicultural practice.

year: 2012