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Title: The Biochemistry of the Latter Stages ofPeptidoglycan Biosynthesis and Modification
Author: Clarke, Thomas Brian
Awarding Body: The University of Warwick
Current Institution: University of Warwick
Date of Award: 2008
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Abstract:
Bacterial cell integrity is maintained by peptidoglycan, a rigid polymer of alternating sugar residues (N-acetlyglucosamine and N-acetylmuramic acid) cross-linked by short peptide stems. Peptidoglycan biosynthesis is a complex three-stage process, under tight spatial and temporal control, with multiple steps exploited as antibiotic targets. The final stage occurs in the periplasm and involves the polymerisation of lipidlI (the final monomeric peptidoglycan precursor) by penicillin-binding proteins, and the attachment of cell surface proteins, containing a C-terminal LPXTG motif, by the sortase family of enzymes. The biochemical characterisation of penicillin-binding proteins and sortases has been mainly limited to investigating interactions with inhibitors and peptide analogues, due to the unavailability of the natural peptidoglycan precursors. Work presented in this thesis describes the development of protocols for the synthesis of both cytoplasmic and lipid-linked peptidoglycan intermediates. It was possible to produce approximately 8S mg of the final cytoplasmic precursor (UDP-MurNAcpentpeptide) in a single-pot incubation. UDP-MurNAc-pentapeptide was then converted to lipidII using a preparation of M jlavlls membranes. Due to the presence of cross~ linking in the peptidoglycan of many Gram-positives, a chemo-enzymatic procedure was used to attach branching amino acids. L-Ala and L-Ala-L-Ala amino acid branches, were attached to the e-amino group of L-Lys in position three of the UDP-MurNAcpentapeptide. These branched derivatives were also converted to lipidII. These substrates were then used to study the enzymology of a variety of pencillin-binding proteins. The transpeptidase activity of two high-molecular weight penicillin-binding proteins (S. aureus PBP2' and E.faecalis PBPS), with intrinsic'low affinity to ~-lactams, was investigated. However, no transpeptidase activity was detected with any monomer substrate. The requirement for the prepolymerisation of the glycan backbone prior to transpeptidation was investigated using two types of polymeric substrate (secreted uncross-linked peptidoglycan polymers and lipidII treated with a monofunctional transglycosylase to polymerise the glycan backbone of lipidII), however, no transpeptidase activity was detected using either of these polymeric substrates. The DDcarboxypeptidase and endopeptidase activity of a low-molecular weight penicillinbinding protein (E. coli PBP4) was investigated with a variety of peptidoglycan fragments. The importance of a small pocket at the base of the E. coli PBP4 transpeptidase active site for substrate recognition was demonstrated by site-direct mutagenesis. The differences in activity of these related classes of enzyme highlights the gap in the understanding of substrate recognition by the transpeptidase domain of pencillin-binding proteins. Sortase enzymes covalently attach proteins to peptidoglycan. Proteins are covalently linked to branched lipidlI, which is subsequently polymerised in to the peptidoglycan polymer. S. pnellmoniae SortaseA was crytsallised, and a fluorescence resonance energy transfer assay was used to detect transpeptidation. This work provides the basis for full structural and biochemical characterisation of Sortase mediated transpeptidation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.502532  DOI: Not available
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