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Enzymatic and structural analyses of PA5540: a gamma-carbonic anhydrase of Pseudomonas aeruginosa PAO1

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dc.contributor.advisor Kimber, Matthew
dc.contributor.author White, Sean
dc.date.accessioned 2014-01-13T20:02:34Z
dc.date.available 2014-12-18T06:00:11Z
dc.date.copyright 2014-01
dc.date.created 2013-12-18
dc.date.issued 2014-01-13
dc.identifier.uri http://hdl.handle.net/10214/7815
dc.description.abstract γ-Carbonic anhydrases are a family of enzymes which catalyze the metal-dependent reversible hydration of carbon dioxide. Based on crystal structures obtained in our lab, we propose that PA5540 of Pseudomonas aeruginosa PAO1 is a novel member of this class. This protein is strongly upregulated in sputum from the lungs of cystic fibrosis patients, suggesting a possible role in pathogenesis. From a biochemical standpoint, PA5540 is intriguing as it is organized in an operon whose genes are only expressed when intracellular zinc is low. This suggests that this ortholog may use a metal other than the zinc as its catalytic metal, with iron and cobalt being candidates. A Pseudomonas putida-based expression system was used to overexpress PA5540 and the ability of PA5540 to act as a functional carbonic anhydrase was tested using a pH indicator dye-based spectrophotometric assay. A maximal observed turnover rate of 660 ± 230 s-1 was obtained at pH 8.3, and a rate of 341± 67 s-1 was obtained at pH 7.5. Due to technical difficulties regarding the assay, true kcat and Km values have not yet been determined. If these values are obtained in the future, atomic absorption spectroscopy can be used to determine and quantify the active site metal. Currently approved sulfonamide drugs are known to inhibit the well characterized γ-carbonic anhydrase, Cam of Methanosarcina thermophila. By demonstrating the activity of PA5540, this research may lead to future methods of combating lung infection, should it be demonstrated that PA5540 contributes to pathogenesis. en_US
dc.description.sponsorship The Natural Sciences and Engineering Research Council of Canada en_US
dc.language.iso en en_US
dc.rights Attribution-NonCommercial-ShareAlike 2.5 Canada *
dc.rights.uri http://creativecommons.org/licenses/by-nc-sa/2.5/ca/ *
dc.subject gamma en_US
dc.subject carbonic en_US
dc.subject anhydrase en_US
dc.subject PA5540 en_US
dc.subject Pseudomonas en_US
dc.subject aeruginosa en_US
dc.subject carbon en_US
dc.subject dioxide en_US
dc.subject bicarbonate en_US
dc.subject CO2 en_US
dc.subject HCO3- en_US
dc.title Enzymatic and structural analyses of PA5540: a gamma-carbonic anhydrase of Pseudomonas aeruginosa PAO1 en_US
dc.type Thesis en_US
dc.degree.programme Molecular and Cellular Biology en_US
dc.degree.name Master of Science en_US
dc.degree.department Department of Molecular and Cellular Biology en_US
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