Abstract
Pseudomonas aeruginosa is an opportunistic bacterium which requires iron for growth and survival. Under iron limiting conditions, P.aeruginosa secrete iron scavenging compounds called pyoverdines. Pyoverdines consist of a chromophore, peptide segment and a side chain. There are three main types of pyoverdine classified depending on iron-chelating peptide component. The peptide region secreted by type I pyoverdine includes an unusual amino acid, formylhydroxyornithine. Formylhydroxyornithine is a conversion product of PvdA, a L-ornithine-N 5 -oxygenase and PvdF, a formyltransferase. Ornithine is converted into hydroxyornithine catalysed by PvdA and hydroxyornithine to formylhydroxyornithine by PvdF. However, little is known about PvdF as an enzyme. The work described in this thesis investigated the structural and functional aspects of PvdF and the interaction of PvdA and PvdF which may facilitate substrate channelling.
A protocol was optimised for purification of hexahistidine tag fused PvdF. Purified PvdF protein was used to carry out structural studies. Secondary structure analysis using CD predicted 44% helices, 23% beta sheets and 33% random coils. Crystallography trials were made but no crystals were obtained. Structure of PvdF was also predicted using ITASSER and showed secondary structure composition consistent with the experimental data obtained by CD analysis. An enzyme assay was developed and PvdF enzyme activity assays were carried out on purified protein. The product, formylhydroxyornithine was detected upon analysis of enzyme reaction mixtures by mass spectrometry. The presence of product indicated that PvdF purified in this study is enzymatically active.
The active site amino acids of PvdF were predicted by superimposing the predicted structure with E. coli GART, a homologous enzyme that catalyzes the conversion of N 1 -(5-phospho-D-ribosyl) glycinamide (GAR) to form N 2 -formyl-N 1 -(5-phospho-D-ribosyl) glycinamide (fGAR). The predicted amino acids were altered by site directed mutagenesis and enzyme activity assays were carried out. D229, N168 and H170 were likely to be catalytic amino acids and G147 to be a binding site amino acid. In cell culture assays, PvdF variants were introduced into Pseudomonas aeruginosa pvdF mutant strains and pyoverdine production assayed. The results were consistent with enzyme activity assay results except that G147A was inactive as pureprotein and active in the cell.
PvdA-PvdF interaction was investigated using Bacterial Adenylate Cyclase Two-hybrid system and protein pull-down assays. BACTH was not able to detect positive interaction between PvdA and PvdF. Pull-down assays using Ni 2+ -resin showed co-purification of hexahistidine tag PvdA and PvdF indicating PvdA-PvdF interaction but hexahistidine tag PvdF was not able to pull-down PvdA. With a C-terminal tag, PvdF was able to pull-down PvdA confirming PvdF-PvdA interaction indicating the hexahistidine tag at the N-terminal of PvdF was interfering PvdA-PvdF interaction. The pull-down assay showed that PvdF with C-terminal hexahistide tag was able to pull- down PvdA confirming hexahistidine tag at N-terminal of PvdF interferes with PvdA-PvdF interaction.
The overall research represents the first structural and functional information of PvdF. The research also showed that PvdA-PvdF form a stable interaction that may facilitate synthesis of formyhydroxyornithine.