Pseudomonas aeruginosa infections constitute a widespread health problem with high economical and social impact, and the
phosphorylcholine phosphatase (
PchP) of this bacterium is a potential target for antimicrobial treatment. However,
drug design requires high-resolution structural information and detailed biophysical knowledge not available for
PchP. An obstacle in the study of
PchP is that current methods for its expression and purification are suboptimal and allowed only a preliminary kinetic characterization of the
enzyme. Herein, we describe a new procedure for the efficient preparation of recombinant
PchP overexpressed in Escherichia coli. The
enzyme is purified from
urea solubilized inclusion bodies and refolded by dialysis. The product of
PchP refolding is a mixture of native
PchP and a kinetically-trapped, alternatively-folded aggregate that is very slowly converted into the native state. The properly folded and fully active
enzyme is isolated from the refolding mixture by size-exclusion chromatography.
PchP prepared by the new procedure was subjected to chemical and biophysical characterization, and its basic optical, hydrodynamic,
metal-binding, and catalytic properties are reported. The unfolding of the
enzyme was also investigated, and its thermal stability was determined. The obtained information should help to compare
PchP with other
phosphatases and to obtain a better understanding of its catalytic mechanism. In addition, preliminary trials showed that
PchP prepared by the new protocol is suitable for crystallization, opening the way for high-resolution studies of the
enzyme structure.