Expression and characterisation of metalloproteins from Mycobacterium tuberculosis
The resurgence of tuberculosis cases world-wide over the last two decades has led to one third of the population being infected and an ever increasing number of deaths (World Health Organisation, 2006). Little is known about the pathogenicity of the infectious agent, Tubercule bacillus, and resistance to the key chemotherapeutic drugs is widespread. Increasing research effort aiming to curtail the spread of this disease has been aided by the work of Cole et al. (1998 and 2002), which provided genomic annotations of the H37Rv strain of Mycobacterium tuberculosis. Subsequent structural genomics projects have identified hundreds of potential targets for structure-based drug design. The research presented in this thesis focuses on the expression and characterisation of targets from the Mycobacterium tuberculosis genome. Cell-free expression trials of 36 unique targets were performed. Initial screening resulted in soluble expression for 30 % of the targets and inclusion of additives, such as molecular chaperones or detergents, increased this to 67 %. Milligram quantities of protein were obtained for eleven targets. As a comparison, four targets were chosen for expression trials using an E. coli in vivo system. Similar results were obtained for three of the targets using the cell-free or in vivo expression systems. However, significant quantities of soluble Rv3545c, a cytochrome P450 125, were only produced using the in vivo method. Proteins that were expressed in sufficient quantities were progressed into crystallisation trials, one of which yielded crystals suitable for X-ray diffraction. The crystal structure of Rv3628, an inorganic pyrophosphatase (Mtb-PPase), was refined to 2.7 A resolution in space group P3221. Inorganic pyrophosphatases (PPases) are ubiquitous metalloenzymes which belong to the phosphatase superfarnily, and play an essential role in biosynthetic reactions (Teplyakov et aL, 1994). 'ne refined crystal structure of Mtb- PPase was found to exhibit a similar overall fold and oligomeric form to existing type I PPase structures. Comparison with two recent Mtb-PPase structures, both in space group P6322 (Tammenkoski et al., 2005 and Benini and Wilson, to be published), highlighted a possible pH-dependent role of His93 within the active site. The characterisation of Rv3545c, a predicted cytochrome P450 125 (Mtb-CYP125), is also described in this thesis. Cytochrome P450s are a superfamily of haern-thiolate proteins (50 to 60 kDa) which monooxygenate hydrophobic substrates as part of electron transport chains (Nebert and Gonzalez, 1987 and Chapple, 1998). P450s have recently been implicated as novel antimycobacterial targets (Munro et al., 2003). Spectroscopy was used to confirm the cytochrome P450 annotation of Rv3545c, with the ferrous enzyme exhibiting a Soret peak at 450 nm in the presence of CO. A high-to-low spin-shift was observed by UV/visible and EPR spectroscopy, upon imidazole-inhibition of ferric Mtb-CYP125. Secondary structural elements were determined by circular dichroism (CD) to be - 33 % a-helix and - 14 % P-sheet. Finally, dark brown/red crystals of Mtb-CYP125 were obtained, but it was not possible to collect a full data set. This was primarily due to the crystals forming clusters which were impossible to separate. Despite this, weak diffraction data to 3 A resolution were measured, and further optimisation of the crystallisation conditions may prove successful.