Enzymes for the homeland defense: optimizing phosphotriesterase for the hydrolysis of organophosphate nerve agents

Abstract

Phosphotriesterase (PTE) from soil bacteria is known for its ability to catalyze the detoxification of organophosphate pesticides and chemical warfare agents. Most of the organophosphate chemical warfare agents are a mixture of two stereoisomers at the phosphorus center, and the S(P)-enantiomers are significantly more toxic than the R(P)-enantiomers. In previous investigations, PTE variants were created through the manipulation of the substrate binding pockets and these mutants were shown to have greater catalytic activities for the detoxification of the more toxic S(P)-enantiomers of nerve agent analogues for GB, GD, GF, VX, and VR than the less toxic R(P)-enantiomers. In this investigation, alternate strategies were employed to discover additional PTE variants with significant improvements in catalytic activities relative to that of the wild-type enzyme. Screening and selection techniques were utilized to isolate PTE variants from randomized libraries and site specific modifications. The catalytic activities of these newly identified PTE variants toward the S(P)-enantiomers of chromophoric analogues of GB, GD, GF, VX, and VR have been improved up to 15000-fold relative to that of the wild-type enzyme. The X-ray crystal structures of the best PTE variants were determined. Characterization of these mutants with the authentic G-type nerve agents has confirmed the expected improvements in catalytic activity against the most toxic enantiomers of GB, GD, and GF. The values of k(cat)/K(m) for the H257Y/L303T (YT) mutant for the hydrolysis of GB, GD, and GF were determined to be 2 × 10(6), 5 × 10(5), and 8 × 10(5) M(-1) s(-1), respectively. The YT mutant is the most proficient enzyme reported thus far for the detoxification of G-type nerve agents. These results support a combinatorial strategy of rational design and directed evolution as a powerful tool for the discovery of more efficient enzymes for the detoxification of organophosphate nerve agents.

Figure 1

Screening of the M317X mutant library against S_p-5 using GWT-d1 as the parental template. The bars in light green, purple and dark blue represent the relative catalytic activity of the GWT-d1, GWT-f1 and GWT-d1-M317F mutants, respectively. Those mutants represented by the gray bars were not characterized or sequenced.

Figure 2

(A) Screening of the 6-site randomized library using GWT-f1 as the parental template with S_p-5. The bars in light green and red represent the relative catalytic activity of the GWT-f1 and GWT-f3 mutants, respectively. (B) Screening of the error-prone PCR library using GWT-f4 as the parental template with S_p-5. The bars in light green, cyan, dark blue, yellow and red represent the GWT-f4, GWT-f4-G129D, GWT-f4-I228F, GWT-f4-G254W and GWT-f5 mutants, respectively.

Figure 3

Outline of the parental lineage for the construction of mutants of PTE that are enhanced for the hydrolysis of S_p-4 and S_p-5.

Figure 4

The localization of the mutations made in QFRN (A), GWT-d3 (B) and GWT-f5 (C). The mutated residues are highlighted in green.

Figure 5

Structural perturbations at the mutated sites in GWT-d3 and GWT-f5. (A) The parent enzyme (GWT) in the region of Lys-185 is highlighted in yellow and the mutant, GWT-d3, is highlighted in cyan. (B) The parent enzyme (GWT) in the region of I274 is highlighted in yellow and the mutant, GWT-d3 is highlighted in cyan. (C) The parent enzyme (GWT) in the region of Ala-80 is highlighted in yellow and the mutant, GWT-f5 is highlighted in cyan. (D) The parent enzyme (GWT) in the region of Arg-67 is highlighted in yellow and the mutant, GWT-f5, is highlighted in green. An imidazole is found in a π–stacking arrangement between the two histidine residues at the subunit interface.

Figure 6

The substrate binding pocket of GWT-d3 (A) and GWT-f5 (B) complexed with compound 7.

Figure 7

Bar graphs illustrating enhanced values for k_cat/K_m (M⁻¹ s⁻¹) for the S_p-enantiomer of compound 1 (A), 2 (B) and 3 (C), S_pR_c-4 (D), S_pS_c-4 (E) and 5 (F) as catalyzed by the wild-type and engineered mutants of PTE.

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Scheme 5