Xylella fastidiosa is a small, xylem-limited bacterium that causes a number of diseases in over 100 species of plants. Many of the species infected are economically important (such as coffee, grapevines, citrus, and almond) and billions of dollars worldwide are lost
annually due to X. fastidiosa infection of crops. The bacterium colonises both plant and insect hosts, using the insect host to transfer it from plant to plant. Sequencing of the X. fastidiosa genome in 2000 discovered that while the genome is reduced, it contains a high
number of putative bifunctional enzymes. One of these enzymes, aspartate kinase:diaminopimelate decarboxylase (AK:DapDc), occurs in only a handful of species and is predicted to catalyse the first and last steps of lysine biosynthesis. This study reports the first experimental characterisation of this enzyme. AK:DapDc was over-expressed in the pET30dSE plasmid in Escherichia coli BL21 DE3 cells. It was purified by Ni2+ His-Trap chromatography followed by size exclusion chromatography. Homology models of AK:DapDc were created in SWISS-MODEL, which indicate homology with the aspartate kinase from Arabidopsis thaliana and the diaminopimelate decarboxylase from E. coli. Circular dichroism, and analytical ultracentrifugation were used to obtain information about the secondary and quaternary structure of AK:DapDc. This data, in combination with the homology models, suggests that AK:DapDc exists as a dimer or tetramer in solution. A
coupled enzyme assay to assay for diaminopimelate decarboxylase activity has been set up, and preliminary crystal screens have been carried out.
| Identifer | oai:union.ndltd.org:canterbury.ac.nz/oai:ir.canterbury.ac.nz:10092/10292 |
| Date | January 2014 |
| Creators | Dorsey, Emma Kathryn |
| Publisher | University of Canterbury. School of Biological Sciences |
| Source Sets | University of Canterbury |
| Language | English |
| Detected Language | English |
| Type | Electronic thesis or dissertation, Text |
| Rights | Copyright Emma Kathryn Dorsey, http://library.canterbury.ac.nz/thesis/etheses_copyright.shtml |
| Relation | NZCU |
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