The intricate workings of an enzyme from Neisseria meningitidis metabolism. (2015)
Type of ContentTheses / Dissertations
Degree NameDoctor of Philosophy
PublisherUniversity of Canterbury
AuthorsHeyes, Logan Colinshow all
3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step in the shikimate pathway, responsible for the biosynthesis of the aromatic amino acids as well as other aromatic metabolites. The reaction involves the aldol like condensation of erythrose 4-phosphate (E4P) and phosphoenol pyruvate (PEP) to form the product DAH7P. The presence of the shikimate pathway in plants, fungi, bacteria and apicomplexan parasites as well as its absence from mammalian cells makes enzymes of the pathway, in particular, the enzyme responsible for the first committed step, useful targets for the design of novel therapeutics. The genome of Neisseria meningitidis expresses a single DAH7PS, thus making it a vulnerable target for drug design. An in depth knowledge of the workings of this enzyme may provide the platform for its efficacy as a target in the fight against bacterial meningitis. Chapter 2 reports the characterisation of three different synthetic inhibitors against NmeDAH7PS and illustrates which of the intermediates formed during the reaction mechanism is the better target. The identification of a single conserved active site water molecule in the active site has implications for the mechanism of NmeDAH7PS and the potency of the synthetic inhibitor. Chapter 3 describes the binding and inhibition of the three aromatic amino acids at the enzyme’s allosteric site. It identifies the reason(s) for the variable sensitivity and gives insight into the inactive conformation adopted by the NmeDAH7PS active site in the presence of an allosteric effector with respect to both substrates. It goes further to identify the effect incurred on substrate binding in the presence of allosteric inhibitor. Chapter 4 investigates the importance of interactions at the tetramer interface for catalysis, regulation, stability and structure of NmeDAH7PS. Two mutations were made, one altering the only salt bridge at the Chain A-C interface and the other involving the only hydrophobic interaction at the Chain A-D interface. Chapter 5 looks into the allosteric binding site and identified two residues that may be crucial for the binding of Phe. The substitution of these two residues and their structural and functional characterisation illustrates the necessity for certain interactions within the largely hydrophobic binding site for binding. This chapter also carries on from work done in chapter 5 on the identification of residues important for the allosteric mechanism. Substitution of these ionisable residues and the structural and functional analysis of the resulting variant enzymes have interesting implications for the electrostatic network responsible for the transfer of allosteric signal in NmeDAH7PS. This thesis gives a detailed description of the inhibitory and regulatory properties of NmeDAH7PS and illustrates the applicability of this work to many other systems in nature.