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
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 forrrfing clusters which were impossible to
separate. Despite this, weak diffraction data to 3 Aresolution were measured, and
further optimisation of the crystallisation conditions may prove successful.