Structure of diethyl phosphate bound to the binuclear metal center of phosphodiesterase

Jungwook Kim, Ping Chuan Tsai, Shi Lu Chen, Fahmi Himo, Steven C. Almo, Frank M. Raushel

Research output: Contribution to journalArticle

52 Citations (Scopus)

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 from the β-metal ion, while the other oxygen is 2.2 Å 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 quantum mechanical/molecular mechanical simulations which postulated the formation of an asymmetrical product complex bound exclusively to the β-metal ion with a metal-metal 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 ́-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.

Original languageEnglish (US)
Pages (from-to)9497-9504
Number of pages8
JournalBiochemistry
Volume47
Issue number36
DOIs
StatePublished - Sep 9 2008

Fingerprint

Phosphoric Triester Hydrolases
Phosphoric Diester Hydrolases
Metals
Metal ions
Hydrolysis
Ions
Cacodylic Acid
Organophosphates
Divalent Cations
Dihydroorotase
Esters
Amidohydrolases
Oxygen
Nucleophiles
Water
Substrates
diethyl phosphate
Density functional theory
Pseudomonas
Protons

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structure of diethyl phosphate bound to the binuclear metal center of phosphodiesterase. / Kim, Jungwook; Tsai, Ping Chuan; Chen, Shi Lu; Himo, Fahmi; Almo, Steven C.; Raushel, Frank M.

In: Biochemistry, Vol. 47, No. 36, 09.09.2008, p. 9497-9504.

Research output: Contribution to journalArticle

Kim, Jungwook ; Tsai, Ping Chuan ; Chen, Shi Lu ; Himo, Fahmi ; Almo, Steven C. ; Raushel, Frank M. / Structure of diethyl phosphate bound to the binuclear metal center of phosphodiesterase. In: Biochemistry. 2008 ; Vol. 47, No. 36. pp. 9497-9504.
@article{32a070b4b327416aab831f7de557afce,
title = "Structure of diethyl phosphate bound to the binuclear metal center of phosphodiesterase",
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 {\AA} from the β-metal ion, while the other oxygen is 2.2 {\AA} from the β-metal ion. The two metal ions are separated by a distance of 4.0 {\AA}. 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 quantum mechanical/molecular mechanical simulations which postulated the formation of an asymmetrical product complex bound exclusively to the β-metal ion with a metal-metal separation of 5.3 {\AA}. 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 ́-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.",
author = "Jungwook Kim and Tsai, {Ping Chuan} and Chen, {Shi Lu} and Fahmi Himo and Almo, {Steven C.} and Raushel, {Frank M.}",
year = "2008",
month = "9",
day = "9",
doi = "10.1021/bi800971v",
language = "English (US)",
volume = "47",
pages = "9497--9504",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "36",

}

TY - JOUR

T1 - Structure of diethyl phosphate bound to the binuclear metal center of phosphodiesterase

AU - Kim, Jungwook

AU - Tsai, Ping Chuan

AU - Chen, Shi Lu

AU - Himo, Fahmi

AU - Almo, Steven C.

AU - Raushel, Frank M.

PY - 2008/9/9

Y1 - 2008/9/9

N2 - 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 from the β-metal ion, while the other oxygen is 2.2 Å 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 quantum mechanical/molecular mechanical simulations which postulated the formation of an asymmetrical product complex bound exclusively to the β-metal ion with a metal-metal 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 ́-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.

AB - 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 from the β-metal ion, while the other oxygen is 2.2 Å 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 quantum mechanical/molecular mechanical simulations which postulated the formation of an asymmetrical product complex bound exclusively to the β-metal ion with a metal-metal 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 ́-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.

UR - http://www.scopus.com/inward/record.url?scp=51549089373&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=51549089373&partnerID=8YFLogxK

U2 - 10.1021/bi800971v

DO - 10.1021/bi800971v

M3 - Article

VL - 47

SP - 9497

EP - 9504

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 36

ER -