Shepherd, Jennifer (2011) Characterisation of pneumococcal peptidoglycan cross-linking enzymology. PhD thesis, University of Warwick.

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Abstract

Despite the introduction of penicillin, infections caused by Streptococcus pneumoniae are associated with significant morbidity and mortality. As a result, there is an urgent need for successful identification of new drug targets within the organism. This thesis focuses on characterisation of MurM, which initiates the synthesis of branched muropeptides within pneumococcal peptidoglycan. MurM and MurN generate either alanyl-alanine or seryl-alanine appendages on the stem peptide lysine of Lipid II, ultimately resulting in indirect cross-linkage of the cell wall. Inactivation of murMN causes a reversion to penicillin sensitivity in penicillinresistant strains. However, elucidation of the relationship between MurM activity and penicillin-resistance is complicated by some penicillin-sensitive strains, including R6, having an unusually high proportion of indirect cross-linkage in their cell wall. Therefore, MurMR6 has been kinetically characterised with Lipid II, AlatRNAAla and Ser-tRNASer for comparison to MurMPn16 (penicillin-sensitive) and MurM159 (penicillin-resistant). These results confirmed that MurM159 is more catalytically active than MurMPn16. However, in the presence of Ser-tRNASer, the catalytic activity of MurMR6 approaches that of MurM159. Stimulation of MurM by cardiolipin indicates the potential role of pneumococcal membrane phospholipid composition in the regulation of this enzyme. Assessment of MurM substrate specificity was made using misaminoacylated SertRNAAla. Results indicate that Ser-tRNAAla is used more efficiently by MurM providing a link between peptidoglycan biosynthesis and the fidelity of protein synthesis in S. pneumoniae. A 2’-amino minihelix analogue of Ala-tRNAAla inhibits MurM with an IC50 of 0.5 μM demonstrating specific acceptance of the amino acid from the 2’ hydroxyl of the terminal adenine of the tRNA substrate. Crystallisation of MurM in the presence of zinc and subsequent characterisation of its metal-ion binding properties by kinetic analysis, isothermal titration calorimetry and bioinformatics-informed site-directed mutagenesis have identified that this enzyme is zinc-dependent. In combination, these findings have far-reaching implications for future drug design.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Streptococcus pneumoniae -- Effect of drugs on, Streptococcus pneumoniae -- Physiology
Date:	September 2011
Institution:	University of Warwick
Theses Department:	School of Life Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Roper, David ; Dowson, Chris
Sponsors:	University of Warwick
Extent:	xxxii, 330 leaves : ill., charts
Language:	eng
URI:	http://wrap.warwick.ac.uk/id/eprint/49184

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