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    Characterisation and Control of 3-Deoxy-D-arabino-heptulosonate 7-phosphate Synthase from Geobacillus sp

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    Othman_M_ thesis_form_2014.pdf (365.5Kb)
    Author
    Othman, Mohamad
    Date
    2014
    Permanent Link
    http://hdl.handle.net/10092/10113
    Thesis Discipline
    Biochemistry
    Degree Grantor
    University of Canterbury
    Degree Level
    Masters
    Degree Name
    Master of Science

    3-Deoxy-D-arabino heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first step of the shikimate pathway, responsible for the biosynthesis of aromatic amino acids. This pathway is found in microorganisms, plants and apicomplexan parasites and its absence in mammals makes it a viable target for antimicrobial drug design. DAH7PS enzymes differ in the regulatory machinery that decorates the catalytic (β/α)8 barrel. Some DAH7PS enzymes are fused to chorismate mutase (CM), another enzyme in the shikimate pathway. This fusion protein is allosterically regulated by chorismate (CA) or prephenate (PA), the precursor of tyrosine and phenylalanine. It has been suggested that DAH7PS enzymes evolved these extensions to the core barrel for the sole purpose of regulation.

    Geobacillus sp DAH7PS (GspDAH7PSWT) is a thermophilic type Iβ DAH7PS enzyme with an N-terminal CM domain fused through a linker region. This thesis describes the functional characterisation work carried out on GspDAH7PSWT, in attempt to help determine how DAH7PS enzymes evolved such diverse methods of regulation.

    Chapter 2 describes the functional characterisation work carried out on the catalytic and regulatory domains of GspDAH7PSWT. The enzyme demonstrated both DAH7PS and CM activities with the DAH7PS domain determined to be metal dependent and most activated by Cd2+. PA completely inhibited the catalytic activity of GspDAH7PSWT, and AUC demonstrated an equilibrium exists between the dimeric and tetrameric quaternary states of the enzyme in solution.

    Chapter 3 describes the domain truncation of GspDAH7PSWT carried out at the linker region in order to obtain two separate protein domains, the catalytic domain lacking the N-terminal domain (GspDAH7PSDAH7PS) and the regulatory domain without the catalytic domain (GspDAH7PSCM). Both variants were fully characterised, and information obtained from each domain was compared to the respective catalytic and regulatory domains of the wild-type enzyme, which was also characterised. Like GspDAH7PSWT, GspDAH7PSDAH7PS showed greatest activation in the presence of Cd2+, with other metals having varying effects on activation rates and stability of the enzyme. Both truncated variants followed Michaelis-Menten kinetics where GspDAH7PSDAH7PS was found to be more active than GspDAH7PSWT and unaffected by PA, whereas GspDAH7PSCM was a less efficient catalyst than the CM domain of GspDAH7PSWT. AUC demonstrated that in solution an equilibrium occurs between the monomeric and tetrameric oligomeric states of GspDAH7PSDAH7PS.

    Chapter 4 summarises the findings of the thesis along with future directions of this research, combining the results obtained and expanding upon them. It is concluded that the catalytic regulatory CM domain supports both protein structure and allosteric regulation of GspDAH7PSWT

    Subjects
    3-Deoxy-D-arabino heptulosonate 7-phosphate synthase
     
    DAH7PS
     
    GspDAH7PS
     
    Geobacillus sp
     
    Gsp
     
    Enzyme
     
    Inhibition
     
    Regulation
     
    Protein
     
    Shikimate Pathway
     
    Chorimate Mutase
     
    CM
     
    Prephenate
     
    Truncation
     
    Crystal
     
    Structure
     
    DSF
     
    Analytical Ultracentrifugation
     
    Metal Activation
     
    Cloning
     
    Purification
     
    Chromatography
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    • Science: Theses and Dissertations [3603]
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