Overexpression, crystallization and preliminary X-ray crystallographic analysis of the phosphotriesterase from Mycobacterium tuberculosis

Abstract

Organophosphates (OPs) are extremely toxic compounds that are used as insecticides or even as chemical warfare agents. Phosphotriesterases (PHPs) are responsible for the detoxification of OPs by catalysing their degradation. Almost 100 PHP structures have been solved to date, yet the crystal structure of the phosphotriesterase from Mycobacterium tuberculosis (mPHP) remains unavailable. This study reports the first crystallization of mPHP. The crystal belonged to space group C222(1), with unit-cell parameters a = 68.03, b = 149.60, c = 74.23 Å, α = β = γ = 90°. An analytical ultracentrifugation experiment suggested that mPHP exists as a dimer in solution, even though one molecule is calculated to be present in the asymmetric unit according to the structural data.

Figure 1

Degradation of the nerve agent sarin by PHPs.

Figure 2

Coomassie Blue-stained 12% SDS–PAGE showing recombinant mPHP before crystallization. Lane M, Protein Ruler I (TransGen Biotech; labelled in kDa). Lane P, recombinant mPHP protein.

Figure 3

Rod-like crystals of mPHP used for data collection at Shanghai Synchrotron Radiation Facility with a wavelength of 1.005 Å.

Figure 4

X-ray diffraction pattern of the mPHP crystal. The circles from the edge to the centre indicate 2, 4, 8, 20 and 40 Å resolution, respectively.

Figure 5

Analytical ultracentrifugation assay for mPHP. The AUC results are presented as a c(M) distribution model.