Investigation of the Roles of Cytoplasmic Enzymes in Zinc Toxicity to the Pathogenic Organism Streptococcus pneumoniae

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Master of Science by Research to the School of Molecular Sciences, College of Science, Health and Engineering, La Trobe University, Victoria, Australia.

Antibiotics have been one of the most important discoveries of the 20th century. However, the misuse of antibiotics has led to the meteoric rise of multidrug resistant pathogens. S. pneumoniae is the leading bacterial cause of pneumonia and primarily infects children under the age of five and adults over the age of 55. According to the World Health Organisation, Streptococcus pneumoniae is considered a medium priority organism for RandD. Due to antibiotic resistance, researchers have recently focused on the antimicrobial effect of zinc against S. pneumoniae. This thesis presents the biochemical and structural characterisation of enzymes that maintain homeostasis within the pathogen (glutathione reductase and NADH oxidase) or are hypothesised to be affected by potential zinc mismetallation (GlmS, GlmM and GlmU). Glutathione reductase was overexpressed, purified and its biochemical properties characterised. The structure of this enzyme was determined by X-ray crystallography to 2.55 a resolution. Michaelis-Menten analyses of the enzyme activity showed that the Km (GSSH) was four times higher than the majority of homologues biochemically analysed. Analysis of NADH oxidase was largely limited to the optimisation of the purification protocol. Analytical ultracentrifugation confirmed NADH oxidase was a dimeric enzyme. Biochemical analysis of GlmS, GlmM and GlmU showed zinc inhibition occurred in the acetyltransferase domain of the bifunctional enzyme, GlmU. The X-ray crystal structure of GlmU bound to zinc was solved to 2.08 Å resolution, identifying Glu-315, His-330 and two H2O molecules as the zinc binding ligands. Analyses of the Zn2 positive-binding properties of the enzyme determined a binding affinity of GlmU for Zn2 positive of 61.8 microM while the GlmUE315H330A mutant enzyme showed no Zn2 positive binding.