Update on biochemical properties of recombinant Pseudomonas diminuta phosphotriesterase.
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. Co(2+)-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 Zn(2+) whereas Co-PTE has Zn(2+) and Co(2+) in equimolar amount, with Co(2+) most likely in the reported labile beta-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 ZnCl(2) induces a non-competitive partial inhibition of Zn(2+)- and Co(2+)-PTE at pH 8.5 (apparent Ki=155 microM and 52 microM, respectively). Inhibition results from interactions with colloidal Zn(OH)(2) formed in alkaline buffer that alters the catalytic machinery. NiCl(2) 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 Co(2+)-PTE, Ki for Zn(OH)(2) inhibition, k(cat) (up to 60 fold) for paraoxon hydrolysis, but not K(M). Issues addressed in this work are important for future biotechnological developments of PTE as a detoxifying enzyme.
Carletti
Eugénie
E
Unité d'Enzymologie, Département de Toxicologie, Centre de Recherches du Service de Santé des Armées, 38700 La Tronche, France.
Jacquamet
Lilian
L
Loiodice
Mélanie
M
Rochu
Daniel
D
Masson
Patrick
P
Nachon
Florian
F
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D015394:Molecular Structure; D044345:Phosphoric Triester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013052:Spectrometry, X-Ray Emission
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D044345:Phosphoric Triester Hydrolases