Badilla Pino, CP;
(2017)
The role of AioX in regulating arsenite oxidation in Rhizobium sp. str NT-26.
Doctoral thesis , UCL (University College London).
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Abstract
Abstract Studies in Rhizobium sp. str. NT-26 have shown that arsenite oxidation is regulated by a two-component signal transduction system comprised of a histidine kinase (AioS) and a response regulator (AioR). A third protein, AioX, is now thought to be involved in the signaling pathway. AioX is a periplasmic binding protein (PBP) whose gene is located upstream and in the same operon as aioS and aioR. The hypothesis is that AioX binds arsenite and activates transcription of the arsenite oxidase (aioB and aioA) operon. The recombinant AioX protein was expressed in Escherichia coli, purified and crystallised. The crystal structure of AioX was solved by single-wavelength anomalous dispersion (SAD) to 1.78 Å resolution and found to contain phosphate in its binding pocket. The structure of AioX shows a similar topology to the PBP’s that consist of two globular regions connected by a hinge, forming a groove between them. The apo-AioX and arsenite-bound structures were also solved by X-ray to a resolution of 1.74 Å and 2.44 Å, respectively. The ligand-binding site of AioX is located at the centre of the protein in a pocket formed between the two domains. The oligomeric state of the protein with and without ligand was analysed by size exclusion chromatography (SEC) and small angle X-ray scattering (SAXS). Isothermal titration calorimetry (ITC) was used to confirm arsenite binding and residues involved in ligand binding were confirmed by site-directed mutagenesis followed by ITC. The thermodynamic properties revealed the recombinant AioX WT had a KD of 170 nM whereas C106S and Y88F mutants showed no binding. A Y88A mutant showed reduced binding with a KD of 1.4 mM. These results suggest that the hydroxyl group of Y88 and C106 are essential in binding arsenite.
| Type: | Thesis (Doctoral) |
|---|---|
| Title: | The role of AioX in regulating arsenite oxidation in Rhizobium sp. str NT-26 |
| Event: | UCL |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1544378 |
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