3URQ

Crystal Structure of PTE mutant H254G/H257W/L303T/M317L/I106C/F132I/L271I/K185R/I274N/A80V/R67H with cyclohexyl methylphosphonate inhibitor

Summary for 3URQ

• Entry DOI: 10.2210/pdb3urq/pdb
• Related: 3UPM 3UR2 3UR3 3URA 3URB 3URN
• Descriptor: Parathion hydrolase, COBALT (II) ION, cyclohexyl methylphosphonate, ... (5 entities in total)
• Functional Keywords: metalloenzyme, tim barrel, nerve agents, phosphotriesterase, hydrolase, hydrolase-hydrolase inhibitor complex, hydrolase/hydrolase inhibitor
• Biological source: Brevundimonas diminuta
• Cellular location: Cell membrane; Peripheral membrane protein: P0A434
• Total number of polymer chains: 2
• Total formula weight: 71792.91

Authors

Tsai, P.,Fox, N.G.,Li, Y.,Barondeau, D.P.,Raushel, F.M. (deposition date: 2011-11-22, release date: 2012-08-01, Last modification date: 2023-12-06)

Primary citation

Tsai, P.C.,Fox, N.,Bigley, A.N.,Harvey, S.P.,Barondeau, D.P.,Raushel, F.M.
Enzymes for the homeland defense: optimizing phosphotriesterase for the hydrolysis of organophosphate nerve agents.
Biochemistry, 51:6463-6475, 2012

PubMed 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.

PubMed: 22809162
DOI: 10.1021/bi300811t

Experimental method

X-RAY DIFFRACTION (2.1 Å)