JOURNAL ARTICLE

Phosphotriesterase variants with high methylphosphonatase activity and strong negative trade-off against phosphotriesters

Luis Briseño-Roa, Christopher M Timperley, Andrew D Griffiths, Alan R Fersht
Protein Engineering, Design & Selection: PEDS 2011, 24 (1-2): 151-9
21037279
The most lethal organophosphorus nerve agents (NA), like sarin, soman, agent-VX and Russian-VX, share a methylphosphonate moiety. Pseudomonas diminuta phosphotriesterase (PTE) catalyses the hydrolysis of methylphosphonate NA analogues with a catalytic efficiency orders of magnitude lower than that towards the pesticide paraoxon. With a view to obtaining PTE variants that more readily accept methylphosphonate NA, ~75,000 PTE variants of the substrate-binding residues Gly-60, Ile-106, Leu-303 and Ser-308 were screened with fluorogenic analogues of the NA Russian-VX and cyclosarin. Seven new PTE variants were isolated, purified and their k(cat)/K(M) determined against five phosphotriesters and five methylphosphonate analogues of sarin, cyclosarin, soman, agent-VX and Russian-VX. The novel PTE variants exhibited as much as a 10-fold increase in activity towards the methylphosphonate compounds--many reaching a k(cat)/K(M) of 10⁶ M⁻¹ s⁻¹--and as much as a 29,000-fold decrease in their phosphotriesterase activity. The mutations found in two of the variants, SS0.5 (G60V/I106L/S308G) and SS4.5 (G60V/I106A/S308G), were modelled into a high-resolution structure of PTE-wild type and docked with analogues of cyclosarin and Russian-VX using Autodock 4.2. The kinetic data and docking simulations suggest that the increase in activity towards the methylphosphonates and the loss of function against the phosphotriesters were due to an alteration of the shape and hydrophobicity of the binding pocket that hinders the productive binding of non-chiral racemic phosphotriesters, yet allows the binding of the highly asymmetric methylphosphonates.

Full Text Links

Find Full Text Links for this Article

Discussion

You are not logged in. Sign Up or Log In to join the discussion.

Trending Papers

Remove bar
Read by QxMD icon Read
21037279
×

Save your favorite articles in one place with a free QxMD account.

×

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"