The proportion of antibiotic resistant Gram-positive strains in the clinic and community continue to rise, despite the number of new antibiotics continuing to fall with time. At the intersection of this problem is the established challenge of working with what has ultimately been both nature’s and humanity’s favoured and most successful antibiotic target, the biosynthesis of the bacterial cell wall. The challenge lies in the predominately membrane/lipid linked habitat that the enzymes and substrates of this complex biosynthetic pathway function within. Membrane protein science remains non-trivial and often difficult, and as such remains undeveloped despite its hugely important role in the medical and biological sciences. As a result, there is a paucity of understanding for this pathway, with limited methods for assay of the activity of the biosynthetic enzymes. These enzyme include the monofunctional transglycosylases, monofunctional transpeptidase penicillin-binding proteins (PBPs) and bifunctional PBPs capable of both transglycosylation and transpeptidation. A number of these enzymes were expressed and purified, with the intention of obtaining novel kinetic and catalytic characterisation of their activities. The more complex of these enzymes could not be proven to be active, and so the comparatively simpler enzyme, an S. aureus monofunctional transglycosylase called MGT, was taken as a model enzyme and used to help design novel assay methods for its transglycosylase activity. The assays developed in this work gave access to novel time-course data and will help demonstrate other interesting mechanistic/catalytic information about the MGT enzyme and of transglycosylation in general. Mathematical modelling was performed around the experimental work. Novel and unique models were designed to define the mechanism of the MGT and generic transglycosylation, as this had not been performed before. The mathematical concepts of structural identifiability and structural indistinguishability were used to analyse these models. Data from experiments were then used to attempt data fitting with the models, and information about the underlying unknown kinetic parameters were collected. Together, a new framework of understanding of the MGT and transglycosylation can be made, which may hopefully be a small step towards answering the challenge now posed by widespread antibiotic resistance.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:582340 |
| Date | January 2012 |
| Creators | Braddick, Darren |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/57211/ |
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