Phosphotriesterases

For these and other reasons, the development of asymmetric catalytic methods for the preparation of enantiopure phosphorus compounds is highly desirable. One approach is to perform hydrolytic kinetic resolution using phosphotriesterases as catalysts (152-154). A typical example is the production of chiral organophosphates (K)-yi or (5)-37. 

It was shown that the phosphotriesterase from P. diminuta catalyzes the enan-tioselective hydrolysis of several racemic phosphates 37, the isomer reacting faster than the compound (153,154). Kinetics experiments showed that the value of is greater for the isomer than for the other by a factor of as much as 

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]. 

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Vilanova E, Sogorb MA. 1999. The role of phosphotriesterases in the detoxication of organophosphoms compounds. Crit Rev Toxicol 29( 1) 21-57. 

Other interesting examples of proteases that exhibit promiscuous behavior are proline dipeptidase from Alteromonas sp. JD6.5, whose original activity is to cleave a dipeptide bond with a prolyl residue at the carboxy terminus [121, 122] and aminopeptidase P (AMPP) from E. coli, which is a prohne-specific peptidase that catalyzes the hydrolysis of N-terminal peptide bonds containing a proline residue [123, 124]. Both enzymes exhibit phosphotriesterase activity. This means that they are capable of catalyzing the reaction that does not exist in nature. It is of particular importance, since they can hydrolyze unnatural substrates - triesters of phosphoric acid and diesters of phosphonic acids - such as organophosphorus pesticides or organophosphoms warfare agents (Scheme 5.25) [125]. 

Phosphotriesterase from P. diminuta (PTE) was found to exhibit high hydrolytic activity towards various types of tetracoordinated phosphorus acid esters. Apart from the phosphonothionate 92, phosphoric acid triesters 94 (Equation 45), °" benzenephosphonic acid diester 95 (Equation 46) ° and methyl-phenylphosphinic acid ester 96 (Equation 47) were also stereoselectively hydrolysed under kinetic resolution conditions. Of course, in the case of the latter three kinds of substrates, half of the reacting ester was irreversibly lost due to the formation of achiral phosphorus acids. 

In the case of the phosphotriesters 94, the use of engineered mutants of phosphotriesterase allowed not only to enhance but even to reverse the stereoselectivity of the native enzyme. The ees of the recovered esters exceeded 95%. 

All the enzymes discussed above belong to the class of dimetalloenzymes. In this context, it should be mentioned that serine-type hydrolases are irreversibly inhibited by organophosphorus esters, among them highly toxic chemical warfare agents. However, in some cases, for example of human butyrylcholi-noesterase, the inhibited enzyme could be reactivated by proper mutations." Moreover, such mutahons were found to confer phosphotriesterase activity in this... 

A search for new enzymes should be continued. Nature proves to be still very unpredictable and sometimes startling discoveries can be made, which are, for example, connected with finding new enzymes of unexpected activity. Phosphotriesterases are the best examples as they catalyse the transformation which is unknown in living systems. 

Mutagenesis of known enzyme towards a desired activity will be the fastest developing direction. The use of mutants of simple serine-hydrolases, which exhibit the phosphotriesterase activity (in contrast to the native enzymes, which are irreversibly inhibited under such conditions), clearly shows that practically any kind of substrates can be enzymatically transformed. The... 

Griffiths, A.D. and Tawfik, D.S. (2003) Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization. The EMBO Journal, 22, 24-35. 

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. 

The inactivation and detoxification of paraoxon and congeners are catalyzed by the so-called A-esterases, which, as discussed, comprise aryleste-rase (sometimes still called paraoxonase, EC 3.1.1.2) and phosphoric triester hydrolases (phosphotriesterases, EC 3.1.8) subdivided into aryldialkylphos-phatase (organophosphate hydrolase, paraoxonase, EC 3.1.8.1) and organophosphorus acid anhydrolases (EC 3.1.8.2 see Sect. 9.3.7) [65][69][106-108], These activities, which occur mostly in the mammalian liver and... 

J. L. Vanhooke, M. M. Benning, F. M. Raushel, H. M. Holden, Three-Dimensional Sducture of the Zinc-Containing Phosphotriesterase with the Bound Substrate Analog Diethyl 4-Methylbenzylphosphonate , Biochemistry 1996, 35, 6020 - 6025. 

LeJeune, K.E., Mesiano, A.J., Bower, S.B., Grismley, J.K., Wild, J.R. and Russell, A.J. (1997) Dramatically stabilized phosphotriesterase-polymers for nerve agent degradation. Biotechnology and Bioengineering, 54(2), 105-114. 

Ghanem E, Raushel EM (2005) Detoxification of organophosphate nerve agents by bacterial phosphotriesterase. Toxicol Appl Pharmacol 207(2, Supplement l) 459-47... 

The mechanism resembles that proposed for a phosphotriesterase (Fig. 12-24). The triesterase catalyzes detoxification of organophosphorus toxins such as parathion (Box 12-E) and seems to have evolved rapidly from a homologous protein of unknown function.721 The phosphotriesterase contains two Zn2+ ions in a dimetal center. An unusual structural feature is a carbamate group, formed from Lys 169 and C02, which provides a bridging ligand for the metal pair.721-725 A carbamylated lysine also functions in ribulose bisphos-phate carboxylase (Fig. 13-11). 

Figure 12-24 Hypothetical event in the action of a phosphotriesterase. A carbamylated lysine (lower center), as well as a water molecule, bridge the two Zn2+ ions, which are held by imidazole and aspartate carboxylate groups. The bound H20 can be deprotonated to give the HO complex shown. The substrate may displace the HO - ion from the right-hand zinc and thereby move close to the bound HO which attacks as indicated. Based on Cd2+-containing structure and discussion by Benning et al.722...

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