RdoA is a bacterial protein kinase from Salmonella enterica serovar Typhimurium first noted for its regulation of dsbA expression in this organism. The crystal structure of RdoA’s homologue, YihE from Escherichia coli, revealed a basic bi-lobal kinase domain that is a hallmark of the eukaryotic Ser/Thr, Tyr protein kinase superfamily. YihE however, bears the greatest structural similarity to choline kinase and aminoglycoside 3’-phosphotransferase [APH(3’)]-IIIa which are both atypical kinases. RdoA and YihE have demonstrated the capacity for autophosphorylation in vitro and the ability to phosphorylate myelin basic protein, however, the native kinase target protein has not been identified. Based on structural alignment with APH(3’)-IIIa, predictions were made of key residues involved in ATP binding and catalysis and five YihE mutants were generated. Both the wildtype and YihE mutants were cloned for expression as N-terminal histidine-tagged proteins. In the work presented here, these proteins have been overexpressed and purified for further study. Mutational analyses revealed that four of the five mutants had decreased kinase activity in comparison to the wildtype protein, thereby establishing the mutated residues as important for enzymatic activity. Several attempts were made to elucidate the substrate of RdoA/YihE, however, it remains unknown. Further investigation is necessary to identify its substrate(s) and to pinpoint its physiological significance. RdoA is a member of the Cpx regulon and its absence stimulates Cpx activation. Since the Cpx system is involved in regulating expression of cell surface appendages and is one of three envelope stress response systems, it is hypothesized that RdoA serves to relay Cpx activation signals. This is supported by studies on the effect of pH on Cpx activity in wildtype and rdoA- cells presented here. RdoA homologues are present in at least 85 different genera. This level of conservation is indicative of an important biological role for this previously uncharacterized bacterial protein kinase. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-12-04 18:19:29.574
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/931 |
| Date | 05 December 2007 |
| Creators | Manu-Boateng, Adwoa |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | 2566842 bytes, application/pdf |
| Rights | This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. |
| Relation | Canadian theses |
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