In Vitro Investigation of the Substrate Specificity of Thiol Dioxygenases
|dc.contributor.advisor||Jameson, Guy N. L.|
|dc.contributor.advisor||Wilbanks, Sigurd M.|
|dc.identifier.citation||Aloi, S. (2016). In Vitro Investigation of the Substrate Specificity of Thiol Dioxygenases (Thesis, Master of Science). University of Otago. Retrieved from http://hdl.handle.net/10523/6650||en|
|dc.description.abstract||Thiol dioxygenases are non-heme mononuclear iron enzymes, which catalyze the oxidation of a thiol to a sulfinic acid. The present study characterized two TDOs from gram-negative bacteria Pseudomonas aeruginosa (p3MDO) and Ralstonia eutropha JMP134 (Re3MDO). The two enzymes are highly similar in their overall cupin-folds, and share a high degree of sequence identity. A detailed kinetic characterization of p3MDO has already been reported, which demonstrated the dual substrate specificity of this enzyme towards 3-mercaptopropionate (3-MPA) and L-cysteine. In all 3-mercaptopropionate dioxygenases (3MDO) a Gln residue equivalent to Arg in CDOs is conserved. The role of this Gln residue in the substrate specificity of 3MDOs was probed in the present study by characterizing a variant at the Gln site of p3MDO. This variant was generated using site-directed mutagenesis. Additionally, the Re3MDO protein was functionally characterized for the first time in the present work, and compared to p3MDO. Enzyme thiol dioxygenation activity was measured by HPLC-ELSD, 1H NMR, and Ellman’s assay. Mass spectrometry was utilized to confirm reaction products. Results showed that the substitution of Gln62 by an Arg residue broadened substrate specificity. The 3MDO activity of Re3MDO was confirmed, and its pH-independent parameters with 3-MPA as substrate were similar to p3MDO. The reactivity of Re3MDO with L-cysteine was negligible.|
|dc.publisher||University of Otago|
|dc.rights||All 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.title||In Vitro Investigation of the Substrate Specificity of Thiol Dioxygenases|
|thesis.degree.name||Master of Science|
|thesis.degree.grantor||University of Otago|
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