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Type: Thesis
Title: Biotin protein ligase as a novel antifungal drug target
Author: Sternicki, Louise Marie
Issue Date: 2020
School/Discipline: School of Biological Sciences
Abstract: Pathogenic fungi are responsible for causing significant disease in both humans and plants. Fungal infections are responsible for 1.5 million deaths annually, and despite the availability of antifungal treatments, mortality rates remain high. Fungi not only cause human disease, but can also infect plants, crops and fruits, thereby, being a major threat for secure food production and the economy. Fungi are developing resistance to the few classes of antifungals that are available to treat human or agricultural infections. Hence, novel antifungal agents are required. Biotin is an essential co-factor required for all forms of life. It is covalently attached to biotin-dependent enzymes and participates in the metabolic reactions these enzymes perform. The enzyme responsible for the ATP-dependent attachment of biotin onto these enzymes is biotin protein ligase (BPL), which attaches biotin onto a conserved lysine residue in the biotin domain of the biotin-dependent enzymes. BPL is essential for the viability of bacteria and fungi, therefore, it is proposed as a promising target for the development of novel anti-infectives. Whilst inhibitors against the BPLs from Staphylococcus aureus and Mycobacterium tuberculosis have been reported, BPL has not been targeted for the development of novel antifungals. This thesis investigates the BPLs from the model fungi Saccharomyces cerevisiae and from two pathogenic fungi responsible for causing agricultural disease. Fungal BPLs are structurally unique compared to bacterial homologs as they contain an additional N-terminal extension prior to the common catalytic domain and C-terminal cap found in all BPLs. BPLs with this extension are termed class III BPLs. Currently there is no atomic resolution structure of a class III enzyme and the function of the additional N-terminal extension is unknown. This thesis investigates the structure of these class III enzymes. The BPLs from Saccharomyces cerevisiae, Botrytis cinerea and Zymoseptoria tritici were characterised for their potential as antifungal drug targets. These three enzymes had similar overall structures as expected from their sequence homology. However, they displayed different stabilities and variable Michaelis constants for the substrates biotin, MgATP and the biotin domain, which implied subtle structural differences between the enzymes. These class III BPLs varied in their preferences for different species’ biotin domains, providing further evidence for a ‘substrate selectivity verification’ mechanism whereby the N-terminal extension of class III BPLs is proposed to distinguish between substrates and select only those appropriate for biotinylation. Finally, potent and selective in vitro inhibition of these class III BPLs was demonstrated using chemical analogs of the reaction intermediate, supporting the hypothesis that fungal BPLs are druggable antifungal targets. The structure of the S. cerevisiae BPL (ScBPL) was investigated in greater detail, and structural changes that occur upon ligand binding were examined. Whilst gross structural changes did not occur concomitant with ligand binding, smaller structural changes including rigidification and a reduction in the dynamic movement of the enzyme occurred such that ScBPL stability increased. Homology modelling and hydrogen-deuterium exchange (HDX) mass spectrometry revealed a structured domain present in the N-terminal extension, with a predicted fold homologous to a glutamine amidotransferase. This N-terminal domain was linked to the structurally conserved BPL catalytic domain by a 160-residue linker that was mostly folded and/or buried. Together, HDX and homology modelling revealed that ligand binding induced local structural rearrangements in the N-terminal domain and the ligand-binding site of the catalytic domain. Finally, HDX also identified potential interaction surfaces of each domain indicating intramolecular interactions that govern BPL function. Overall, this project aims to understand the structure of a class III BPL in order to target these enzymes for the development of novel antifungal agents for the treatment of both agricultural and clinical infections.
Advisor: Booker, Grant
Wegener, Kate
Polyak, Steven
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2020
Keywords: Biotin protein ligase
structural biology
native Mass Spectrometry
Provenance: This thesis is currently under Embargo and not available.
Appears in Collections:Research Theses

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