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    Investigations into the Inhibition of 3-Deoxy-D-manno-Octulosonate 8-Phosphate Synthase

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    Author
    Harrison, Aidan Nicholas
    Date
    2010
    Permanent Link
    http://hdl.handle.net/10092/5382
    Thesis Discipline
    Chemistry
    Degree Grantor
    University of Canterbury
    Degree Level
    Doctoral
    Degree Name
    Doctor of Philosophy

    The enzyme 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyses the aldol condensation of the five-carbon sugar phosphate, arabinose 5-phosphate (A5P), and phosphoenol pyruvate (PEP) to give the eight-carbon phosphorylated sugar, KDO8P. It is the second committed step in the synthesis of KDO, a necessary component of the cell wall of Gram-negative bacteria. This thesis describes the design, synthesis and evaluation of a number of inhibitors of KDO8P synthase that utilise the functionality of one or both substrates. The KDO8P synthase family can be divided based on the requirement of a divalent metal ion. Chapter 2 describes the growth, purification and characterisation of an example from both the metal-independent KDO8P synthases (Neisseria meningitidis, Nme) and metal-dependent KDO8P synthases (Acidithiobacillus ferrooxidans, Afe) in order to utilise these enzymes for the inhibition studies described in this thesis. In Chapter 3, a number of small molecule PEP analogues were selected as mimics of KDO8P synthase reaction intermediates and tested as inhibitors of KDO8P synthase from N. meningitidis and A. ferrooxidans. Glyphosate, (E)-vinyl phosphonate and the fluorinated analogue of (E)-vinyl phosphonate were selected as mimics of the high-energy oxocarbenium intermediate through which the KDO8P synthase reaction is thought to occur. The two enantiomers of phospholactate were selected in order to investigate the chirality of the tetrahedral intermediate and determine the importance of this chirality for inhibition of KDO8P synthase. All five inhibitors were found to be moderate to poor inhibitors of both the KDO8P synthase from N. meningitidis and A. ferrooxidans. Chapter 4 describes the design and synthesis of inhibitors that incorporated structural features of the second substrate, A5P, in order to improve inhibition from that observed for the PEP analogues investigated in Chapter 3. A bisphosphate inhibitor was designed that incorporated a terminal phosphate moiety, representative of the phosphate of A5P. A large increase in inhibition was found, compared to the phospholactates from which it was derived. A structure-activity-relationship study was undertaken on this compound by design of compounds that lacked one of the two phosphate moieties of the bisphosphate inhibitor, in order to determine their relative importance. The inhibition results indicate that the primary terminal phosphate, thought to bind in the A5P phosphate binding site, is more important for inhibition of KDO8P synthase than the secondary phosphate. In Chapter 5 these investigations into the inhibition of KDO8P synthase are discussed in detail, and interpreted using the aid of computational studies. In addition several approaches are described for the completion and advancement of the studies presented here in this thesis.

    Subjects
    KDO8P synthase
     
    KDO8PS
     
    3-Deoxy-D-manno-Octulosonate 8-Phosphate Synthase
     
    Inhibitor
     
    enzyme
     
    chemistry
     
    Gram-negative bacteria
    Collections
    • Science: Theses and Dissertations [3298]
    Rights
    http://library.canterbury.ac.nz/thesis/etheses_copyright.shtml

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