Investigating the structure, function and inhibition of DHDPS from an intracellular pathogen

Abstract

© 2014 Tanzeela SiddiquiEnzymes of the diaminopimelate (DAP) pathway have attracted much attention in the past two decades as potential antimicrobial targets. The end products of this pathway, meso-DAP and lysine, are essential components in bacterial cell walls and protein synthesis. One key enzyme catalysing the rate-limiting step in the DAP pathway is dihydrodipicolinate synthase (DHDPS). DHDPS has been extensively characterised from plant and bacterial species. However, little information is available on the enzyme from intracellular bacteria. As such, this thesis examines the structure and function of DHDPS from the intracellular, Gram-negative pathogen Legionella pneumophila. DHDPS from L. pneumophila was cloned from gDNA, followed by expression and purification of recombinant protein to homogeneity. Identity of the purified product was confirmed using mass spectrometry and the overall fold was similar to a classical DHDPS enzyme, as determined by CD spectroscopy. Kinetic analyses also showed that L. pneumophila DHDPS functioned in a similar capacity to E. coli DHDPS, with a kcat of 101 s-1, KM of 0.24±0.01 mM for pyruvate and KM of 0.19±0.01 mM for (S)-ASA. At the commencement of this research project, DHDPS enzymes existed predominantly as homotetramers. The formation of DHDPS tetramers is thought to facilitate catalysis by restricting movement of key active site residues. Through use of X-ray crystallography, small angle X-ray scattering and analytical ultracentrifugation, it is shown that L. pneumophila DHDPS forms the less frequently observed dimeric structure. Examination of the 1.65 Å L. pneumophila DHDPS crystal structure reveals a greater number of contacts at the interface between both DHDPS monomers relative to the E. coli homolog. This lends weight to the theory of an alternate evolutionary solution for limiting flexibility of active site residues, as previously proposed for the MRSA DHDPS dimer. Work presented in this dissertation also challenges the currently accepted paradigm of allosteric inhibition in DHDPS enzymes. To date, all plant and Gram-negative DHDPS respond to feedback inhibition by lysine. A key finding of the work described here is the lack of allosteric regulation displayed by L. pneumophila DHDPS. This represents the first example of a DHDPS enzyme from a Gram-negative pathogen to remain insensitive to lysine inhibition. Examination of the allosteric site in the crystal structure shows substitutions of key lysine-binding residues. An overlay of the DHDPS allosteric sites from L. pneumophila and E. coli reveal these substitutions to either hinder the binding of lysine or prevent formation of critical bonds with lysine. Importantly, the observed lack of inhibition may be linked to the intracellular lifestyle of L. pneumophila. A high demand for meso-DAP and lysine in this bacterium, coupled to their low availability in human cells, likely reflects the absence of regulation in L. pneumophila DHDPS. Alternatively, L. pneumophila DHDPS may be regulated by other means. Availability of high resolution X-ray data for the L. pneumophila DHDPS structure enables visibility of two conformations of the critical catalytic residue Tyr106. The conformation with higher occupancy is within hydrogen-bonding distance of other key active site residues. In contrast, the positioning of the lower occupant reveals disruption to a vital proton relay. This suggests a previously unidentified mechanism for DHDPS inhibition that may replace allosteric regulation in intracellular forms of the enzyme. Findings from this work are expected to broaden knowledge of key sites within the enzyme, thereby aiding in the future design of inhibitor molecules against DHDPS. Further development of such molecules may lead to a new class of antimicrobials that will help combat the impending issue of antibiotic resistance in bacteria