title: Cysteine biosynthesis and the role of CysE in Neisseria gonorrhoeae

authors: Oldham, Keely

abstract: Neisseria gonorrhoeae is the causative organism of the sexually transmitted infection (STI), gonorrhoea. Over decades of antibiotic use, the emergence of antibiotic-resistant strains of N. gonorrhoeae has dramatically increased and we are facing the reality of almost untreatable gonorrhoea. Combined with increasing incidence, there is an urgent need for new antimicrobial treatments for gonorrhoea infection. The synthesis of the amino acid cysteine is a promising new target for the development of new antimicrobials. Cysteine plays an important role in protein molecules, but also in the synthesis of glutathione for protection against oxidative stress. In addition, cysteine is the key step for the incorporation of sulphur into a variety of cellular constituents. N. gonorrhoeae displays unique differences in assimilation of sulphate for the synthesis of cysteine, yet little is known about the synthesis of this important amino acid in N. gonorrhoeae. 

The first step in the dual-step cysteine biosynthesis pathway is catalysed by the serine acetyltransferase, CysE, an essential gene in N. gonorrhoeae. To design inhibitors against this key enzyme we need to elucidate the enzymatic mechanism and the three-dimensional structure. In this thesis, we determine the kinetic parameters and regulation of CysE and present the structure of CysE to 2.01 Å. Biochemical assays demonstrate that CysE has serine acetyltransferase activity and experiences substrate inhibition by acetyl CoA. CysE is also sensitive to feedback inhibition by L-cysteine, which competitively inhibits CysE relative to the substrate L-serine. The structure shows CysE belongs to the left-handed β helix family and adopts a hexameric structure consisting of a dimer of trimers. Our structure reveals the first evidence of domain swapping for a CysE structure, where C-terminal tails domain swap with neighbouring monomers in the crystal lattice. Although an artefact of crystallisation it provides insight into the flexibility of this region. 

Collectively the data presented in this thesis represents key advances in our understanding of the uncharacterised cysteine biosynthetic pathway in N. gonorrhoeae. Data presented here is the basis for future work using the structure of CysE to guide computational inhibitor design, to identify lead compounds for CysE inhibition, and for the development of new antimicrobials for treatment of gonorrhoea.

text: Cysteine biosynthesis and the role of CysE in Neisseria gonorrhoeae Neisseria gonorrhoeae is the causative organism of the sexually transmitted infection (STI), gonorrhoea. Over decades of antibiotic use, the emergence of antibiotic-resistant strains of N. gonorrhoeae has dramatically increased and we are facing the reality of almost untreatable gonorrhoea. Combined with increasing incidence, there is an urgent need for new antimicrobial treatments for gonorrhoea infection. The synthesis of the amino acid cysteine is a promising new target for the development of new antimicrobials. Cysteine plays an important role in protein molecules, but also in the synthesis of glutathione for protection against oxidative stress. In addition, cysteine is the key step for the incorporation of sulphur into a variety of cellular constituents. N. gonorrhoeae displays unique differences in assimilation of sulphate for the synthesis of cysteine, yet little is known about the synthesis of this important amino acid in N. gonorrhoeae. 

The first step in the dual-step cysteine biosynthesis pathway is catalysed by the serine acetyltransferase, CysE, an essential gene in N. gonorrhoeae. To design inhibitors against this key enzyme we need to elucidate the enzymatic mechanism and the three-dimensional structure. In this thesis, we determine the kinetic parameters and regulation of CysE and present the structure of CysE to 2.01 Å. Biochemical assays demonstrate that CysE has serine acetyltransferase activity and experiences substrate inhibition by acetyl CoA. CysE is also sensitive to feedback inhibition by L-cysteine, which competitively inhibits CysE relative to the substrate L-serine. The structure shows CysE belongs to the left-handed β helix family and adopts a hexameric structure consisting of a dimer of trimers. Our structure reveals the first evidence of domain swapping for a CysE structure, where C-terminal tails domain swap with neighbouring monomers in the crystal lattice. Although an artefact of crystallisation it provides insight into the flexibility of this region. 

Collectively the data presented in this thesis represents key advances in our understanding of the uncharacterised cysteine biosynthetic pathway in N. gonorrhoeae. Data presented here is the basis for future work using the structure of CysE to guide computational inhibitor design, to identify lead compounds for CysE inhibition, and for the development of new antimicrobials for treatment of gonorrhoea.

year: 2020

title: A map of the nanoworld: Sizing up the science, politics and business of the infinitesimal

authors: Munshi, Debashish

subjects: nanotechnology

abstract: Mapping out the eight main nodes of nanotechnology discourse that have emerged in the past decade, we explore how various scientific, social, and ethical islands of discussion have developed, been recognized, and are being continually renegotiated. We do so by (1) identifying the ways in which scientists, policy makers, entrepreneurs, educators, and environmental groups draw boundaries on issues relating to nanotechnology; (2) describing concisely the perspectives from which these boundaries are drawn; and (3) exploring how boundaries on nanotechnology are marked and negotiated through contestations of power among various nodes of nanotechnology discourse.

text: A map of the nanoworld: Sizing up the science, politics and business of the infinitesimal Mapping out the eight main nodes of nanotechnology discourse that have emerged in the past decade, we explore how various scientific, social, and ethical islands of discussion have developed, been recognized, and are being continually renegotiated. We do so by (1) identifying the ways in which scientists, policy makers, entrepreneurs, educators, and environmental groups draw boundaries on issues relating to nanotechnology; (2) describing concisely the perspectives from which these boundaries are drawn; and (3) exploring how boundaries on nanotechnology are marked and negotiated through contestations of power among various nodes of nanotechnology discourse.

year: 2007

title: The timing of the human discovery and colonization of New Zealand

authors: Sutton, Douglas G.

abstract: In discriminating between the proposed Long, Short and Intermediate Chronologies for New Zealand prehistory, archaeologists have recently voted by a narrow majority that the human commensal, Rattus exulans, was present in New Zealand up to 2000 years BP, although the preferred date of ‘first settlement’ by people was ca 800 years BP. A recalibration of New Zealand chronometric data has resulted in reinterpretations of the date and duration of occupation on critical archaeological sites. A remodelling of the ages of the putatively earliest sites in New Zealand has shown these to indicate only a broadly defined period when settlement occurred rather than the date when New Zealand was discovered (as the Short Chronology advocates have insisted). Finally, comparisons with archaeological research on islands elsewhere in the world have brought with them recognition that the discovery, exploration, initial settlement and colonization of New Zealand by Polynesian voyagers were broadly consecutive processes. This paper reviews a range of current evidence in the context of its current theorization and affirms the Long Chronology, recognizing it as the most plausible hypothesis.

text: The timing of the human discovery and colonization of New Zealand In discriminating between the proposed Long, Short and Intermediate Chronologies for New Zealand prehistory, archaeologists have recently voted by a narrow majority that the human commensal, Rattus exulans, was present in New Zealand up to 2000 years BP, although the preferred date of ‘first settlement’ by people was ca 800 years BP. A recalibration of New Zealand chronometric data has resulted in reinterpretations of the date and duration of occupation on critical archaeological sites. A remodelling of the ages of the putatively earliest sites in New Zealand has shown these to indicate only a broadly defined period when settlement occurred rather than the date when New Zealand was discovered (as the Short Chronology advocates have insisted). Finally, comparisons with archaeological research on islands elsewhere in the world have brought with them recognition that the discovery, exploration, initial settlement and colonization of New Zealand by Polynesian voyagers were broadly consecutive processes. This paper reviews a range of current evidence in the context of its current theorization and affirms the Long Chronology, recognizing it as the most plausible hypothesis.

year: 2008