Structure of Diethyl Phosphate Bound to the Binuclear Metal Center of Phosphotriesterase

Abstract

The bacterial phosphotriesterase (PTE) from Pseudomonas diminuta catalyzes the hydrolysis of organophosphate esters at rates close to the diffusion limit. X-ray diffraction studies have shown that a binuclear metal center is positioned in the active site of PTE and that this complex is responsible for the activation of the nucleophilic water from solvent. In this paper the three dimensional structure of PTE was determined in the presence of the hydrolysis product, diethyl phosphate (DEP), and a product analogue, cacodylate. In the structure of the PTE-diethyl phosphate complex the DEP product is found symmetrically bridging the two divalent cations. The DEP displaces the hydroxide from solvent that normally bridges the two divalent cations in structures determined in the presence or absence of substrate analogues. One of the phosphoryl oxygen atoms in the PTE-DEP complex is 2.0 Å away from the α-metal ion while the other oxygen is 2.2 Å away from the β-metal ion. The two metal ions are separated by a distance of 4.0 Å. A similar structure is observed in the presence of cacodylate. Analogous complexes have previously been observed for the product complexes of isoaspartyl dipeptidase, D-aminoacylase, and dihydroorotase from the amidohydrolase superfamily of enzymes. The experimentally determined structure of the PTE-diethyl phosphate product complex is inconsistent with a recent proposal based upon QM/MM simulations which postulated the formation of an asymmetrical product complex bound exclusively to the β-metal ion with a metalmetal separation of 5.3 Å. This structure is also inconsistent with a chemical mechanism for substrate hydrolysis that utilizes the bridging hydroxide as a base to abstract a proton from a water molecule loosely associated with the α-metal ion. Density functional theory (DFT) calculations support a reaction mechanism that utilizes the bridging hydroxide as the direct nucleophile in the hydrolysis of organophosphate esters by PTE. † This work was supported in part by the NIH (GM71790 and GM68550) and the Robert A. Welch Foundation (A-840). JK was supported by GM76988. Supporting information available Electron density for cacodylate bound to the active site of G60A ( Figure S1) and for diethyl phosphate bound to wild type PTE ( Figures S2A and S2B). This material is available free of charge via the Internet at

Molecular dynamics simulations of the detoxification of paraoxon catalyzed by phosphotriesterase

“…But Chen et al .’s high level QM optimizations on reduced cluster models led to a similar intermediate state 36. Our result is further supported by the most recent crystallographic structure of the Co/Co-PTE-diethyl phosphate complex (PDB code: 3CAK), where the diethyl phosphate (DEP) complex symmetrically bridges the two Co 2+ ions 62. This structure shows similarities with the structure I1 .…”

Substitution of the Catalytic Metal and Protein PEGylation Enhances Activity and Stability of Bacterial Phosphotriesterase

“…The catalytic mechanism of PTE involves concerted action of two ion complexes of Zn 2+ (or Co 2+ ), and the metal ion complex acts as an electrophilic activator of water molecules. The substrate interaction with the active site of PTE increases the electrophilicity of the organophosphate polarizing the P0O or P0S bonds, making them more susceptible to hydroxyl attack [20].…”

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Theoretical study of the hydrolysis mechanism of 2-pyrone-4,6-dicarboxylate (PDC) catalyzed by LigI

“…These questions cannot be acquired by experiments alone. Therefore, in the present work, the catalytic mechanism of LigI was further studied by using a combined quantum mechanics and molecule mechanics (QM/MM) method, which has been successfully applied in exploring the enzymatic mechanism in the past years [17][18][19][20][21][22][23][24]. Based on the results of our calculations, the energetic details of the whole reaction cycle have been given, the structures of the reactant and its involved species, and the roles of key residues have been delineated at atomistic level.…”