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Title: Mechanistic studies of E. coli YdiA : an ADP-dependent phosphotransferase
Author: Hollins , Jeffrey John
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2012
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PDRP is an unusual bifunctional ADP-dependent phosphotransferase involved in the regulation of C4 plant photosynthesis. Despite thirty years of research since its discovery, biophysical, kinetic, and structural characterisations have remained elusive. The discovery of a homologous protein (YdiA) in E. coli provides an unparalleled opportunity to obtain such characterisations. For the first time, YdiA has been cloned, overexpressed, and the biophysical characteristics have been determined. The protein was found to be dimeric by gel filtration, native-PAGE, and AUC. ITC was used to characterise binding affinity for the putative substrates. The protein binds in a largely temperature and pH-independent manner to ADP, AMP, phosphate, and pyrophosphate, in the uM range, with KdS of \.24 ± 0. 76, 5.23 ± l.l0, 15.54 ± 4.30, and 24.18 ± 2.70 respectively. The putative macromolecular substrate, PPS, was cloned, overexpressed, and characterised. This substrate was found to be dimeric at high concentration by gel filtration, and monomeric at low concentration by native-PAGE. YdiA was shown to interact with PPS in the presence of ATP, by gel filtration. The biophysical data for this interaction could not be obtained. Initial data using peptidcs howcver suggest that YdiA could bind to PtsI. To determine the structure of YdiA, mutagenesis, bioinformatics, and X-ray crystallography were employed; however, it was not possible to determine the structure. Finally, two new coupled assays were developed to obtain a kinetic characterisation of YdiA. This involved the cloning, overexpression, and characterisation of five genes and four proteins.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available