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Structural characterisation of biotin protein ligase and its interacting partners: a novel antibiotic

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thesis
posted on 23.02.2017, 03:07 by Yap, Min Yin
Biotin protein ligase (BPL) is an enzyme found in all organisms, and is required for the attachment of biotin to a specific lysine residue located on the biotin carboxyl carrier protein (BCCP) domain of biotin-dependent metabolic carboxylases which are involved in a variety of metabolic processes which include lipogenesis, amino acid catabolism and gluconeogenesis. BPL can be divided into three main classes: the first 2 classes consist of BPLs found in bacteria, class I bacterial BPLs consist of a C-terminal cap domain and catalytic domain, but not a DNA-binding domain, whilst class II bacterial BPLs have an extra N-terminal DNA binding domain which is used to repress the transcription of biotin biosynthetic genes. Eukaryotic BPLs fall into class III, and whilst their C-terminal cap domain and catalytic domain have sequence similarity to their bacterial counterparts, they also contain a Nterminal domain which does not bind to DNA, and to date its function and structure is still under investigation. Bacterial BPL is a novel target as an antibacterial, due to it being essential for bacterial cell survival. Chapters 3 and 4 of this thesis consist of two published journal articles, and focus on the design of antibacterials that specifically target the BPL from S. aureus (SaBPL), a pathogen which is one of the main causes of mortality in hospitals and in the community, and has rapidly gained resistance to many modern antibiotics. In chapter 5, the expression, purification and preliminary characterization of K. pneumoniae and A. calcoaceticus BPL was performed, as an initial step for future drug design studies. Class II BPLs have to regulate two vastly different roles, the biotinylation of biotin-dependent enzymes, as well as binding to the bioO operator to repress biotin synthesis. The work in chapter 6 uses a combination of X-ray crystallography and small angle X-ray scattering (SAXS) to provide structural information of how the class II BPL, SaBPL binds to its binding partners, BCCP and bioO. This chapter includes a recently published paper as well as further experiments done to obtain a protein crystals of the SaBPL:bioO complex. As the structure of eukaryotic BPLs have not yet been determined, the final chapter of this thesis focuses on the expression and purification of the human BPL, holocarboxylase synthetase (HCS) for use in future structural studies. Structural information not only provides information into the function of HCS, but would also aid in our drug design studies as outlined in chapters 3 to 5.

History

Principal supervisor

Matthew Wilce

Year of Award

2014

Department, School or Centre

Biochemistry and Molecular Biology

Campus location

Australia

Faculty

Faculty of Medicine Nursing and Health Sciences