Braddick, Darren (2012) Quantitative assay methods and mathematical modelling of peptidoglycan transglycosylation. PhD thesis, University of Warwick.

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Abstract

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.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QD Chemistry Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Chemistry, Analytic -- Quantitative, Enzymes -- Analysis, Peptidoglycans -- Analysis, Gram-positive bacteria, Glycosylation -- Mathematical models
Official Date:	September 2012
Dates:	Date Event September 2012 Submitted
Institution:	University of Warwick
Theses Department:	Department of Chemistry; School of Engineering; Systems Biology Doctoral Training Centre
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Bugg, Tim; Chappell, M. J. (Michael J.)
Extent:	xviii, 235 leaves : illustrations, map, charts.
Language:	eng

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