Structural determinants for the stereoselective hydrolysis of chiral substrates by phosphotriesterase

Ping Chuan Tsai, Yubo Fan, Jungwook Kim, Lijiang Yang, Steven C. Almo, Yi Qin Gao, Frank M. Raushel

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Wild-type phosphotriesterase (PTE) preferentially hydrolyzes the R P enantiomers of the nerve agents sarin (GB) and cyclosarin (GF) and their chromophoric analogues. The active site of PTE can be subdivided into three binding pockets that have been denoted as the small, large, and leaving group pockets based on high-resolution crystal structures. The sizes and shapes of these pockets dictate the substrate specificity and stereoselectivity for catalysis. Mutants of PTE that exhibit substantial changes in substrate specificity and the ability to differentiate between chiral substrates have been prepared. For example, the G60A mutant is stereoselective for the hydrolysis of the RP enantiomer of the chromophoric analogues of sarin and cyclosarin, whereas the H254G/H257W/L303T (GWT) mutant reverses the stereoselectivity for the enantiomers of these two compounds. Molecular dynamics simulations and high-resolution X-ray structures identified the correlations between structural changes in the active site and the experimentally determined kinetic parameters for substrate hydrolysis. New high-resolution structures were determined for the H257Y/L303T (YT), I106G/F132G/H257Y (GGY), and H254Q/H257F (QF) mutants of PTE. Molecular dynamics calculations were conducted using the SP and RP enantiomers of the analogues for sarin and cyclosarin for the wild-type PTE and the G60A, YT, GGY, QF, and GWT mutants. The experimental stereoselectivity correlated nicely with the difference in the computed angle of attack for the nucleophilic hydroxide relative to the phenolic leaving group of the substrate.

Original languageEnglish (US)
Pages (from-to)7988-7997
Number of pages10
JournalBiochemistry
Volume49
Issue number37
DOIs
StatePublished - Sep 21 2010

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Phosphoric Triester Hydrolases
Enantiomers
Sarin
Hydrolysis
Stereoselectivity
Substrates
Molecular Dynamics Simulation
Substrate Specificity
Molecular dynamics
Catalytic Domain
Angle of attack
Catalysis
Kinetic parameters
Crystal structure
X-Rays
X rays
Computer simulation
cyclohexyl methylphosphonofluoridate

ASJC Scopus subject areas

  • Biochemistry
  • Medicine(all)

Cite this

Structural determinants for the stereoselective hydrolysis of chiral substrates by phosphotriesterase. / Tsai, Ping Chuan; Fan, Yubo; Kim, Jungwook; Yang, Lijiang; Almo, Steven C.; Gao, Yi Qin; Raushel, Frank M.

In: Biochemistry, Vol. 49, No. 37, 21.09.2010, p. 7988-7997.

Research output: Contribution to journalArticle

Tsai, Ping Chuan ; Fan, Yubo ; Kim, Jungwook ; Yang, Lijiang ; Almo, Steven C. ; Gao, Yi Qin ; Raushel, Frank M. / Structural determinants for the stereoselective hydrolysis of chiral substrates by phosphotriesterase. In: Biochemistry. 2010 ; Vol. 49, No. 37. pp. 7988-7997.
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AU - Tsai, Ping Chuan

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AU - Almo, Steven C.

AU - Gao, Yi Qin

AU - Raushel, Frank M.

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N2 - Wild-type phosphotriesterase (PTE) preferentially hydrolyzes the R P enantiomers of the nerve agents sarin (GB) and cyclosarin (GF) and their chromophoric analogues. The active site of PTE can be subdivided into three binding pockets that have been denoted as the small, large, and leaving group pockets based on high-resolution crystal structures. The sizes and shapes of these pockets dictate the substrate specificity and stereoselectivity for catalysis. Mutants of PTE that exhibit substantial changes in substrate specificity and the ability to differentiate between chiral substrates have been prepared. For example, the G60A mutant is stereoselective for the hydrolysis of the RP enantiomer of the chromophoric analogues of sarin and cyclosarin, whereas the H254G/H257W/L303T (GWT) mutant reverses the stereoselectivity for the enantiomers of these two compounds. Molecular dynamics simulations and high-resolution X-ray structures identified the correlations between structural changes in the active site and the experimentally determined kinetic parameters for substrate hydrolysis. New high-resolution structures were determined for the H257Y/L303T (YT), I106G/F132G/H257Y (GGY), and H254Q/H257F (QF) mutants of PTE. Molecular dynamics calculations were conducted using the SP and RP enantiomers of the analogues for sarin and cyclosarin for the wild-type PTE and the G60A, YT, GGY, QF, and GWT mutants. The experimental stereoselectivity correlated nicely with the difference in the computed angle of attack for the nucleophilic hydroxide relative to the phenolic leaving group of the substrate.

AB - Wild-type phosphotriesterase (PTE) preferentially hydrolyzes the R P enantiomers of the nerve agents sarin (GB) and cyclosarin (GF) and their chromophoric analogues. The active site of PTE can be subdivided into three binding pockets that have been denoted as the small, large, and leaving group pockets based on high-resolution crystal structures. The sizes and shapes of these pockets dictate the substrate specificity and stereoselectivity for catalysis. Mutants of PTE that exhibit substantial changes in substrate specificity and the ability to differentiate between chiral substrates have been prepared. For example, the G60A mutant is stereoselective for the hydrolysis of the RP enantiomer of the chromophoric analogues of sarin and cyclosarin, whereas the H254G/H257W/L303T (GWT) mutant reverses the stereoselectivity for the enantiomers of these two compounds. Molecular dynamics simulations and high-resolution X-ray structures identified the correlations between structural changes in the active site and the experimentally determined kinetic parameters for substrate hydrolysis. New high-resolution structures were determined for the H257Y/L303T (YT), I106G/F132G/H257Y (GGY), and H254Q/H257F (QF) mutants of PTE. Molecular dynamics calculations were conducted using the SP and RP enantiomers of the analogues for sarin and cyclosarin for the wild-type PTE and the G60A, YT, GGY, QF, and GWT mutants. The experimental stereoselectivity correlated nicely with the difference in the computed angle of attack for the nucleophilic hydroxide relative to the phenolic leaving group of the substrate.

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