dc.contributor.author Keith, Alasdair Donald dc.date.accessioned 2022-01-26T00:23:18Z dc.date.available 2022-01-26T00:23:18Z dc.date.issued 2022-05-20 dc.date.submitted 2021-09-01 dc.identifier.uri https://www.repository.cam.ac.uk/handle/1810/332914 dc.description.abstract Many pathogens function by internalising the haem molecules of their host organism and breaking down the porphyrin scaffold to sequester the Fe$^{2+}$ ion. Typically, this breakdown mechanism is mediated by a haem oxygenase. However, a novel class of reaction has been discovered, which can be performed anaerobically using 'nature's reductant', NADH, and the $\textit{Yersinia enterocolitica}$ protein, HemS. To study the features of this reaction in more detail, conventional experimental methods were combined with Energy Landscape Theory. Deuterium labelling demonstrated that the reaction was initiated by hydride transfer and stopped-flow spectroscopy showed that the reaction proceeded $\textit{via}$ a short-lived intermediate. Since no structural information regarding NADH-binding to HemS was available, computational calculations were used to sample the conformational space around possible NADH-protein binding sites and to construct kinetic transition networks. From these networks, pathways showing the unfolding and approach of NADH to haem inside the pocket were determined. These pathways highlighted the roles of various residues, thus allowing for a targeted mutagenesis study. This study, carried out using both computation and laboratory-based experimentation, was especially focussed on a double phenylalanine gate located in the centre of the main cavity. Key insight concerning how this feature regulates the access of NADH to haem was gained. Computational results suggested that the HemS homologues, HmuS, ChuS and ShuS, were also capable of promoting anaerobic haem breakdown, but that catalysis by ChuS and ShuS may be limited by competing functions. Bioinformatics was used to gauge what these possible alternative functions could be, and to place HemS within its wider phylogenetic context. The computational predictions were then tested in the laboratory. The three homologues were all shown to engage in the reductive haem breakdown process but to varying degrees of efficacy. These findings demonstrate that this novel haem breakdown reaction is not unique to HemS, but instead is a feature of a wider class of haemoproteins. A subset of these haemoproteins are known to bind certain DNA promoter regions, suggesting not only that they can catalytically degrade haem, but that they are also involved in transcriptional modulation responding to haem flux. Many of the bacterial species responsible for this class of protein (including those that produce HemS, ChuS and ShuS) are known to specifically target oxygen-depleted regions of the gastrointestinal tract. A deeper understanding of anaerobic haem breakdown processes engaged in by these pathogens could therefore prove useful in the development of future strategies for disease prevention. dc.description.sponsorship Cambridge Trust Vice-Chancellor's Award; Departmental Funding dc.rights All Rights Reserved dc.rights.uri https://www.rioxx.net/licenses/all-rights-reserved/ dc.subject Haem dc.subject Biophysical Chemistry dc.subject Molecular Biology dc.subject Energy Landscapes dc.subject Protein Structure dc.title Anaerobic, NADH-Dependent Haem Breakdown in a Family of Haemoproteins dc.type Thesis dc.type.qualificationlevel Doctoral dc.type.qualificationname Doctor of Philosophy (PhD) dc.publisher.institution University of Cambridge dc.date.updated 2022-01-24T23:27:59Z dc.identifier.doi 10.17863/CAM.80344 rioxxterms.licenseref.uri https://www.rioxx.net/licenses/all-rights-reserved/ rioxxterms.type Thesis dc.publisher.college Jesus pubs.funder-project-id EPSRC (1944583) cam.supervisor Wales, David cam.supervisor Barker, Paul cam.supervisor.orcid Wales, David [0000-0002-3555-6645] cam.depositDate 2022-01-24 pubs.licence-identifier apollo-deposit-licence-2-1 pubs.licence-display-name Apollo Repository Deposit Licence Agreement
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