Update on biochemical properties of recombinant Pseudomonas diminuta phosphotriesterase

Abstract

Phosphotriesterase from Pseudomonas diminuta (PTE; EC 3.1.8.1) hydrolyzes organophosphate insecticides and chemical warfare agents. The two zinc cations in the active center can be substituted. Co2+-containing PTE is the most efficient but least stable isoform. Gel filtration showed that PTE is monomeric at the submicromolar concentrations used in kinetic assays. The analysis of the recombinant enzyme by X-ray fluorescence spectrometry and CCT-ICP-MS, confirms that recombinant Zn-PTE contains only Zn2+ whereas Co-PTE has Zn2+ and Co2+ in equimolar amount, with Co2+ most likely in the reported labile β-site. We noted that recombinant PTE is unstable at low concentrations and must be stabilized by a protein environment. We tested the effect of excess of various metal cofactors on PTE-catalyzed hydrolysis of paraoxon. We notably observed that ZnCl2 induces a non-competitive partial inhibition of Zn2+- and Co2+-PTE at pH 8.5 (apparent Ki=155 μM and 52 μM, respectively). Inhibition results from interactions with colloidal Zn(OH)2 formed in alkaline buffer that alters the catalytic machinery. NiCl2 caused a similar effect at higher concentrations (apparent Ki=3 mM). We observed that mutating His123, a surface residue close to an alleged allosteric site, dramatically altered the bacterial expression yield of Co2+-PTE, Ki for Zn(OH)2 inhibition, kcat (up to 60 fold) for paraoxon hydrolysis, but not KM. Issues addressed in this work are important for future biotechnological developments of PTE as a detoxifying enzyme.