Studies towards the selective inhibition of β-alanine pathways in Mycobacterium tuberculosis
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.