Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.816597
Title: Biophysical studies on enzymes related to antibiotics
Author: Kamps, Jos J. A. G.
ISNI:       0000 0004 9355 3682
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2020
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Abstract:
The work described in this thesis concerns two research areas: (i) time resolved (tr) crystallography studies on two enzymes, oxacillinase 10 (OXA-10) and isopenicillin N synthase (IPNS); and (ii) the reactivity of formaldehyde towards amino acids. OXA-10 is a class D serine β-lactamase that confers bacterial resistance against clinically relevant β-lactam antibiotics. IPNS is a non-heme Fe dependent enzyme that catalyses the formation of isopenicillin N (IPN) from L-δ-(α-aminoadipoyl)-L-cysteinyl D-valine (ACV), which is an important step in the biosynthesis of penicillins and cephalosporins. Formaldehyde is an environmental pollutant and a metabolite in many biochemical reactions. Formaldehyde is toxic and considered carcinogenic above threshold levels, however its reactivity with biomolecules is not well defined. Chapter 1 provides an overview of the literature regarding the biosynthesis of pencillins and cephalosporins and emphasizes the β-lactam ring formation step catalysed by IPNS, discussing the proposed catalytic mechanism in more detail. The role of β-lactamases, and in particular OXA-10, in antibiotic resistance is also discussed. Chapter 2 provides an introduction to tr-crystallography, with an emphasis on the use of X-ray free electron lasers (XFELs). A brief description of the physics behind XFELs is given, as well as the application of XFELs in macromolecular crystallography. Preparation, characterisation, and sample delivery of microcrystal slurries for serial crystallography is introduced. Chapter 3 describes methods for the preparations of OXA-10, and discusses the results of (tr-)crystallographic studies on OXA-10. Different crystallisation conditions for OXA-10 were explored and probed for diffraction using either synchrotron or XFEL radiation. A combination of fixed target and drop-on-demand sample delivery systems were used for the serial crystallography studies on OXA-10. Chapter 4 describes the preparation of ACV, IPNS, and a 19F labelled IPNS S55C variant. The chapter covers the use of (tr-)crystallographic studies on IPNS. IPNS was co-crystallised to give IPNS • Fe • ACV microcrystals, which were exposed to O2 in order to initiate the reaction, and probed for diffraction at defined time points using XFEL radiation. Diffraction data and X-ray emission spectroscopy data were recorded in parallel. The results demonstrated a conformational change in an α-helix and β-strand region and suggested that Fe leaves the active site of IPNS after IPN formation in crystallo. To monitor the conformational changes in an α-helix and β-strand region of IPNS a 19F labelled IPNS S55C variant was produced and used for 19F NMR experiments. The results provided evidence for similar conformational changes of the α-helix and β-strand region in solution, consistent with the time resolved crystallographic studies. Non-denaturing mass spectrometry and 19F NMR studies were used to investigate the loss of Fe at the IPNS active site after IPN formation, as observed in crystallo. The results suggest that IPN induced loss of Fe occurs in crystallo but not in solution. Chapter 5 summarises the work conducted on OXA-10 and IPNS, and discusses lessons drawn from the experimental work described. Chapter 6 provides an overview of the literature concerning the exposure of humans to exogenous and endogenous sources of formaldehyde and summarizes the effects of formaldehyde on health. Previous biochemical and chemical studies on the reactivity of formaldehyde with biomolecules (e. g. DNA, proteins, peptides, nucleobases, amino acids) are discussed. Chapter 7 describes studies on the reactions of formaldehyde with amino acids as monitored using NMR spectroscopy. The products of the reactions were characterised, and their relative stabilities investigated. Competition assays using different amino acids were carried out to establish their relative reactivities. Different biologically relevant carbonyl containing molecules were tested and their reactivities compared with that of formaldehyde. The results demonstrate that formaldehyde was more reactive and forms more stable adducts with amino acids than the other tested compounds.
Supervisor: Schofield, Chris ; Claridge, Tim Sponsor: Clarendon Fund ; Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.816597  DOI: Not available
Keywords: Chemistry
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