Structure-function relationships of the biotin transporters from Staphylococcus aureus
| dc.contributor.advisor | Booker, Grant William | |
| dc.contributor.advisor | Polyak, Steven William | |
| dc.contributor.author | Azhar, Al | |
| dc.contributor.school | School of Biological Sciences | en |
| dc.date.issued | 2015 | |
| dc.description.abstract | The clinically relevant human pathogen Staphylococcus aureus employs an energy coupling factor (ECF) transporter to import the important micronutrient biotin. Like the well characterised ABC transporters, the ECF transporters utilise the hydrolysis of ATP to move substrates across biological membranes. However, the ECF transporters do not use a solute-binding protein to bind substrate instead employing a membrane embedded protein to fulfil this role. In certain bacteria, the substrate binding protein required for binding biotin is known as BioY. The aim of this thesis was to investigate protein structure and function relationships involving the BioY protein from S. aureus (SaBioY). S. aureus has functional import and export processes that result in a biphasic profile of biotin uptake. Active translocation of biotin was temperature-dependent, optimum at 30 minutes, and inhibited by biotin or structural analogues of the vitamin. The study demonstrated that recombinant SaBioY could be expressed in E. coli, and was localised to the membrane fraction as observed by Western blot analysis on fractionated cell lysates and fluorescence microscopy. Importantly, a convenient ligand binding assay was developed that facilitated deeper analysis of the SaBioY structure and function. SaBioY primarily recognises the ureido ring of biotin for substrate capture, but an intact thiophene ring also aids binding. Although a variety of functional groups can be appended onto the carboxyl group of the biotin moiety, the linker used to connect the molecules and the chemical property of functional group can impact binding to SaBioY. This knowledge can be exploited for developing biotin-based analogues with applications in antibiotic drug discovery. Since an X-crystal structure of SaBioY is not available, membrane topology predictions and computational modelling were used to generate a molecular module of SaBioY. This yielded a model containing 5 transmembrane domains, 3 extracellular loops, and 1 intracellular loop with intracellular N- and C-termini. Whilst the model was in good agreement with known crystal structures of other known S components, it possessed an additional V-shaped membrane embedded helix. Conserved amino acid residues in BioY were identified using the web-based Clustal-W alignment program and then mapped onto the SaBioY model. A series of 24 SaBioY mutants were then generated using random and site-directed mutagenesis approaches. Fluorescence polarisation based competitive-binding assays using a fluorescent-biotin tracer revealed several conserved (R75, D157 and K160) and non-conserved (N38, T54, F81, F88 and D128) residues important for biotin binding. Interestingly, a double mutant D157K/K160E completely abolished biotin binding. A filter disk diffusion assay using a panel of antibiotics showed recombinant expression of SaBioY increased E. coli antibiotic sensitivity to streptomycin, erythromycin and chloramphenicol, probably by forming a pore through channel by dimerisation requiring a dynamic cooperative interaction. Expression of all of the SaBioY mutants increased sensitivity to the three antibiotics. The D157K/K160E double mutant was an exception, as it had no effect upon antibiotic sensitivity. We proposed that D157 and K160 together play an essential role in SaBioY activity. In conclusion, this study successfully characterised the SaBioY transporter in both its native state and using a recombinant E. coli expression system. Substrate specificity of the transporter was determined, as was the channel gating potency of SaBioY for certain antibiotics when expressed in E. coli. Computational modelling and a novel FP based competitive assay also provided useful tools for biochemical analysis of SaBioY structure and function relationships. Further studies are now required to determine the SaBioY X-ray crystal structure, transport mechanism and regulation as well as to explore possible application of the transporter as a novel drug target or an alternative gating system new antibiotic agents. | en |
| dc.description.dissertation | Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2015. | en |
| dc.identifier.doi | 10.4225/55/582d34f83333e | |
| dc.identifier.uri | http://hdl.handle.net/2440/102580 | |
| dc.provenance | This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at: http://www.adelaide.edu.au/legals | en |
| dc.subject | BioY | en |
| dc.subject | biotin | en |
| dc.subject | ECF transporter | en |
| dc.subject | recombinant | en |
| dc.subject | fluorescent polarisation | en |
| dc.title | Structure-function relationships of the biotin transporters from Staphylococcus aureus | en |
| dc.type | Theses | en |
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