Thesis (MSc (Chemistry and Polymer Science))--University of Stellenbosch, 2006. / The focus of this study was the pathways for β-alanine production in Mycobacterium
tuberculosis (Mtb), the causative agent of tuberculosis. The major pathway for β-alanine
production is the decarboxylation of L-aspartate by L-aspartate-α-decarboxylase (PanD).
This enzyme is not essential for the survival for Mtb which implies that an alternative
pathway for β-alanine production must exist. We postulated that such a secondary
pathway may be based on the oxidation of various polyamines by a polyamine oxidase to
give the β-alanine precursor 3-aminopropanal, and therefore set out to find data in
support of this hypothesis.
Based on sequence homology to the FAD-dependent Saccharomyces cerevisiae polyamine
oxidase Fms1, Mtb AofH was identified as a likely candidate. The soluble expression and
purification of AofH proved troublesome and lead to the investigation of various
techniques to increase protein yield. These methods include fusion to various tags, coexpression
with the protein chaperones, addition of scarce codon tRNA’s to the
translation mixture and protein refolding. AofH was eventually purified as fusions to the
Nus and MBP proteins and its activity determined by analysis of the enzymatic reactions
by TLC, reverse phase HPLC, ESI-MS and LC-MS. TLC analysis indicated that 3-
aminopropanol formed as a product during polyamine oxidation, but this could not be
confirmed by any of the more sensitive analytical techniques. We set out to confirm the
presence of the FAD cofactor in the enzyme by various methods and concluded that the
AofH fusions did not contain FAD. Efforts to refold the protein in the presence of FAD
also failed. From this study it is clear that the biochemical confirmation of the presumed
activity of AofH will remain elusive until the enzyme can be purified in its active form,
i.e. with FAD bound. A genetic test for activity based on functional complementation
studies of Escherichia coli ΔpanD strains proved inconclusive since no difference in growth
rate was found between cell transformed with the aofH gene and the negative control.
We continued our studies of β-alanine biosynthesis by attempting the design of
mechanism-based inhibitors for the PanD enzyme. Various structural analogues were
identified and tested by qualitative and quantitative methods. Our results show that β-
substituted aspartate analogues may be good potential inhibitors of Mtb’s PanD protein
and can thus be used in rational drug design.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/1596 |
| Date | 03 1900 |
| Creators | Koekemoer, Lizbe |
| Contributors | Strauss, E., Jardine, M. A., University of Stellenbosch. Faculty of Science. Dept. of Chemistry and Polymer Science. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 2369488 bytes, application/pdf |
| Rights | University of Stellenbosch |
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