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dc.contributor.advisorHicks, Joanna
dc.contributor.authorMcGarvie, Jack
dc.date.accessioned2021-10-12T02:03:46Z
dc.date.available2021-10-12T02:03:46Z
dc.date.issued2021
dc.identifier.citationMcGarvie, J. (2021). Structural and biochemical characterisation of O-acetylserine sulphydrylase (CysK) from Neisseria gonorrhoeae (Thesis, Master of Science (Research) (MSc(Research))). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/14596en
dc.identifier.urihttps://hdl.handle.net/10289/14596
dc.description.abstractNeisseria gonorrhoeae is the causative bacteria of the sexually transmitted infection (STI), gonorrhoea. Rapid emergence of antibiotic resistant strains of N. gonorrhoeae has given rise to almost untreatable gonorrhoea over the last few decades. There is an urgent need for development of new antimicrobial treatments for gonorrhoea infection. Synthesis of the amino acid cysteine is a promising new target for the development of new antimicrobials. Cysteine plays a vital role in protein folding and function, and synthesis of glutathione for protection against oxidative stress during infection. The final step of the two-step cysteine biosynthesis pathway is catalysed by the enzyme O-acetylserine sulphydrylase (OASS). Most bacteria have two isoforms of OASS, OASS-A/CysK that utilises sulphide and O-acetylserine for the synthesis of cysteine and OASS-B/CysM that utilises thiosulphate. N. gonorrhoeae has just one OASS isoform (annotated as CysK) in its genome. N. gonorrhoeae displays unique differences in its sulphate acquisition pathway for cysteine biosynthesis, yet little is known about these pathways in N. gonorrhoeae. We propose CysK is a potential antimicrobial target. This thesis biochemically characterises the structure and function of CysK to determine its role in cysteine biosynthesis. Kinetic characterisation demonstrates that CysK has O-acetylserine sulphydrylase activity and displays positive cooperativity with respect to substrates, O-acetylserine and sulphide. Sulphide shows partial allosteric inhibition, and the enzyme does not use thiosulphate as a substrate. The structure of CysK was solved to 2.49 Å by X-ray crystallography and shows CysK belongs to the tryptophan synthase β superfamily and adopts a homodimeric structure consisting of two monomers. The structure supports positive cooperativity as co-factor binding residues are in inactive and active conformations in each monomer of the dimer, respectively. Many CysK enzymes form complexes with the first enzyme in the two-step cysteine synthesis pathway, CysE. It appears CysE and CysK from N. gonorrhoeae are unable to form a complex, indicating an alternate pathway for regulation of sulphur flux and cysteine production. Attempts to investigate the in vivo role of CysK were inconclusive. The research presented in this thesis represents a major leap in our understanding of the uncharacterised cysteine biosynthesis pathway in N. gonorrhoeae. Data presented here is the basis for future work using the kinetics and structure of CysK to guide computational inhibitor design, to identify lead compounds for CysK inhibition, and thereby development of new antimicrobials for treatment of extensively drug resistant gonorrhoea.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.publisherThe University of Waikato
dc.rightsAll items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectBiochemistry
dc.subjectEnzyme kinetics
dc.subjectCrystal structure
dc.subjectCysK
dc.subjectCysteine biosynthesis
dc.subjectCysE
dc.subjectCysteine synthase complex
dc.subject.lcshNeisseria gonorrhoeae -- Treatment
dc.subject.lcshAnti-infective agents
dc.subject.lcshCysteine
dc.subject.lcshEnzyme inhibitors
dc.subject.lcshAmino acids -- Synthesis
dc.titleStructural and biochemical characterisation of O-acetylserine sulphydrylase (CysK) from Neisseria gonorrhoeae
dc.typeThesis
thesis.degree.grantorThe University of Waikato
thesis.degree.levelMasters
thesis.degree.nameMaster of Science (Research) (MSc(Research))
dc.date.updated2021-10-09T02:35:35Z
pubs.place-of-publicationHamilton, New Zealanden_NZ


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