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dc.contributor.advisorJameson, Guy N. L.
dc.contributor.advisorWilbanks, Sigurd M.
dc.contributor.authorFellner, Matthias
dc.date.available2015-05-28T21:16:58Z
dc.date.copyright2015
dc.identifier.citationFellner, M. (2015). Enzymology and Structure of the Thiol Dioxygenase from Pseudomonas aeruginosa (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/5685en
dc.identifier.urihttp://hdl.handle.net/10523/5685
dc.description.abstractThiol dioxygenases catalyse the oxidation of a thiol substrate with dioxygen to produce a sulfinic acid. The mammalian thiol dioxygenase cysteine dioxygenase (CDO), is specific to its substrate L-cysteine, and has been investigated for more than 40 years. However, during the last decade additional thiol dioxygenases have been discovered and all showed a high specificity for their substrate. In-depth kinetic and structural characterisation of an additional thiol dioxygenase was undertaken to support a comparative analysis of the structural basis of thiol dioxygenase activity and substrate specificity. This thesis presents the first extensive characterisation of a putative bacterial CDO homologue from the eubacterium Pseudomonas aeruginosa. For determination of kinetic parameters a new activity assay was developed. This new assay utilises a 96 well plate format and is therefore much more time efficient compared to available techniques. The depletion of any thiol substrate by a thiol dioxygenase can be studied quickly and easily without compromising data quality, however with the drawback that product formation has to be investigated with another method in parallel. For the thiol dioxygenase P. aeruginosa kinetic parameters (kcat and Km) for two different substrates (3-mercaptopropionic acid and L-cysteine) were gathered across a broad pH range, providing a fuller picture than for any thiol dioxygenase other than mammalian CDO. The results will contribute to predicting future putative thiol dioxygenase function as key differences in the sequence of putative CDOs have been compared to experimental findings. The findings show that thiol dioxygenases can have a broad substrate profile and a short list of the protein residues responsible for different substrate specificity of thiol dioxygenases is proposed. Kinetic studies were performed in tandem with X-ray crystallographic characterisation of the thiol dioxygenase structure; this combination is a first for a putative bacterial CDO homologue. The second sphere protein residues appear to be rotated in reference to the active site iron when compared with mammalian CDO. The structure also offers a new insight into an intensively researched feature of CDO, a conserved cysteine-tyrosine crosslink. The thiol dioxygenase from P. aeruginosa lacks this post-translational modification but the active site environment appears to be conserved. This was further proven by structural characterisation of single point mutation (G95C), resulting in the first artificially introduced crosslink into a naturally uncrosslinked thiol dioxygenase. However this mutation dramatically decreased enzymatic activity while having minimal affect on the structure. In addition, crystal structures of single point mutations (C93G, Y157F) abolishing the crosslink in mammalian CDO were also solved. These structures are the first crystal structures of mammalian CDO lacking the crosslink. Comparison of all crystal structures shows that the crosslink does not serve a general stabilisation purpose. The work shows that the protein possesses a dual substrate profile making it a true thiol dioxygenase, it will therefore be referred to P. aeruginosa thiol dioxygenase – pTDO.
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectenzyme
dc.subjectprotein
dc.subjectdioxygenase
dc.subjectthiol
dc.subjectcrystallography
dc.subjectkinetics
dc.subjectenzymology
dc.subjectstructure
dc.subjectPseudomonas aeruginosa
dc.subjectiron
dc.titleEnzymology and Structure of the Thiol Dioxygenase from Pseudomonas aeruginosa
dc.typeThesis
dc.date.updated2015-05-28T08:42:37Z
dc.language.rfc3066en
thesis.degree.disciplineChemistry
thesis.degree.nameDoctor of Philosophy
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral
otago.interloanno
otago.openaccessAbstract Only
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