Disrupting the quaternary structure of DHDPS as a new approach to antibiotic design

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dc.contributor.advisor Gerrard, JA en
dc.contributor.advisor Jameson, GB en
dc.contributor.advisor Devenish, SR en
dc.contributor.advisor Perugini, MA en
dc.contributor.advisor Parker, EJ en
dc.contributor.author Evans, Genevieve en
dc.date.accessioned 2012-04-01T21:58:43Z en
dc.date.issued 2010-01 en
dc.identifier.citation Sub type: PhD Thesis. Supervisors: Gerrard JA, Jameson GB, Devenish SR, Perugini MA, Parker EJ. University of Canterbury, Christchurch, New Zealand, Jan 2010 en
dc.identifier.uri http://hdl.handle.net/2292/16281 en
dc.description.abstract Doctor of Philosophy in Biochemistry Disrupting the quaternary structure of DHDPS as a new approach to antibiotic design G. L. Evans School of Biological Sciences, University of Canterbury, Christchurch, New Zealand This thesis examined the enzyme dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) from the pathogen Mycobacterium tuberculosis. DHDPS is a validated antibiotic target for which no potent inhibitor based on substrates, intermediates or product has been found. The importance of the homotetrameric quaternary structure in E. coli DHDPS has been demonstrated by the 100-fold decrease in activity observed in a dimeric variant, DHDPS-L197Y, created by site-directed mutagenesis. This suggested a new approach for inhibitor design: targeting the dimer-dimer interface and disrupting tetramer formation. DHDPS catalyzes the first committed step in the biosynthetic pathway of meso-diaminopimelic acid, a critical component of the mycobacterial cell wall. In this study, wild-type M. tuberculosis DHDPS was thoroughly characterized and compared with the E. coli enzyme. A coupled assay was used to obtain the kinetic parameters for M. tuberculosis DHDPS: KM(S) ASA = 0.43 (±0.02) mM, KMpyruvate = 0.17 (±0.01) mM, and kcat = 138 (±2) s 1. Biophysical techniques showed M. tuberculosis DHDPS to exist as a tetramer in solution. This is consistent with the crystal structure deposited as PDB entry 1XXX. The crystal structure of M. tuberculosis DHDPS showed active-site architecture analogous to E. coli DHDPS and a dimeric variant of M. tuberculosis DHDPS was predicted to have reduced enzyme activity. A dimeric variant of M. tuberculosis DHDPS was engineered through a rationally designed mutation to analyze the effect of disrupting quaternary structure on enzyme function. A single point mutation resulted in a variant, DHDPS-A204R, with disrupted quaternary structure, as determined by analytical ultracentrifugation and gel-filtration chromatography. DHDPS-A204R was found to exist in a concentration-dependent monomer-dimer equilibrium, shifted towards dimer by the presence of pyruvate, the first substrate that binds to the enzyme. The secondary and tertiary structure of DHDPS-A204R was analogous to wild-type M. tuberculosis DHDPS as judged by circular dichroism spectroscopy and X ray crystallography, respectively. Surprisingly, this disrupted interface mutant had similar activity to the wild type enzyme, with a kcat of 119 (±6) s-1; although, the affinity for its substrates were decreased: KM(S) ASA = 1.1 (±0.1) mM, KMpyruvate = 0.33 (±0.03) mM. These results indicated that disruption of tetramer formation does not provide an alternative direction for drug design for DHDPS from M. tuberculosis. Comparison with the recently discovered dimeric DHDPS from Staphylococcus aureus shed further light on the role of quaternary structure in DHDPS. In M. tuberculosis DHDPS-A204R and the naturally dimeric enzyme, the association of monomers into the dimer involves a greater buried surface area and number of residues than found in E. coli DHDPS-L197Y. This provides a framework to discriminate which DHDPS enzymes are likely to be inactive as dimers and will direct future work targeting the dimer-dimer interface of DHDPS as an approach for drug design. en
dc.publisher University of Canterbury. Biological Sciences en
dc.relation.ispartof PhD Thesis - University of Auckland en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher. en
dc.rights.uri https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm en
dc.title Disrupting the quaternary structure of DHDPS as a new approach to antibiotic design en
dc.type Thesis en
thesis.degree.grantor University of Canterbury, Christchurch, New Zealand en
thesis.degree.level Doctoral en
thesis.degree.name PhD en
dc.rights.holder Copyright: The author en
pubs.author-url http://ir.canterbury.ac.nz/handle/10092/4469 en
dc.rights.accessrights http://purl.org/eprint/accessRights/RestrictedAccess en
pubs.elements-id 190530 en
pubs.record-created-at-source-date 2010-12-03 en


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