Phosphotriesterases Pseudomonas diminuta

The phosphotriesterase from Pseudomonas diminuta was shown to catalyze the enantioselective hydrolysis of several racemic phosphates (21), the Sp isomer reacting faster than the Rp compound [65,66]. Further improvements using directed evolution were achieved by first carrying out a restricted alanine-scan [67] (i.e. at predetermined amino acid positions alanine was introduced). Whenever an effect on activity/ enantioselectivity was observed, the position was defined as a hot spot. Subsequently, randomization at several hot spots was performed, which led to the identification of several highly (S)- or (R)-selective mutants [66]. A similar procedure was applied to the generation of mutant phosphotriesterases as catalysts in the kinetic resolution of racemic phosphonates [68]. 

D.P. Dumas, H.D. Durst, W.G. Landis, F.M. Raushel, and J.R. Wild, Inactivation of organophosphorus nerve agents by the phosphotriesterase from Pseudomonas diminuta. Arch. Biochem. Biophys. 277, 155-159 (1990). 

K.I. Dave, C.E. Miller, and J.R. Wild, Characterization of. organophosphorous hydrolases and the genetic manipulation of. the phosphotriesterase from pseudomonas diminuta. Chem. Biol. Intract. 87, 55-68 (1993). 

A phosphotriesterase isolated from the soil bacterium Pseudomonas diminuta is the best characterized enzyme of this type. There is evidence for the presence of two active site Zn2+ ions in vivo. A crystal structure of the dinuclear Cd2+ form is available in which the metal ions are bridged by a carbamylated Lys-amino group with a metal-metal distance of 3.8 A [ 18]. Substrate hydrolysis follows a SN2 type reaction and nucleophilic attack of M-OH is likely, but mechanistic details are not yet clear. 

Caldwell SR, Raushel FM. Detoxification of organophosphate pesticides using an immobilized phosphotriesterase from Pseudomonas diminuta. Biotechnol Bioeng 1991 37 103-109. 

Carletti, E., Jacquamet, L., Loiodice, M., Rochu, D., Masson, P., Nachon, F. (2009). Update on biochemical properties of recombinant Pseudomonas diminuta phosphotriesterase. J. Enz. Inhib. Med. Chem. (In press)... 

The phosphotriesterase from the soil bacterium Pseudomonas diminuta is the only one that has been well characterized. The natural substrate for this enzyme is unknown it hydrolyzes a large number of phosphotriester substrates, but the best substrate is paraoxon.244 Phosphomonoesters are not substrates, and diesters are hydrolyzed only at greatly reduced rates.245... 

Dumas, D.P, J.R. Wild, and FM. Raushel. 1989. Diisopropylfluorophosphate hydrolysis by a phosphotriesterase from Pseudomonas diminuta.. Appl. Biotech. 11 235-243. 

Dumas DP, Durst HD, Landis WG, Raushel FM, Wild JR (1990) Inactivation of organo-phosphorus nerve agents by the phosphotriesterase from Pseudomonas diminuta. Arch Biochem Biophys 227 155-159... 

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