Functional annotation and three-dimensional structure of Dr0930 from deinococcus radiodurans, a close relative of phosphotriesterase in the amidohydrolase superfamily

Dao Feng Xiang, Peter Kolb, Alexander A. Fedorov, Monika M. Meier, Lena V. Fedorov, Tinh T. Nguyen, Reinhard Sterner, Steven C. Almo, Brian K. Shoichet, Frank M. Raushel

Research output: Contribution to journalArticle

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Abstract

Dr0930, a member of the amidohydrolase superfamily in Deinococcus radiodurans, was cloned, expressed, and purified to homogeneity. The enzyme crystallized in the space group P3 121, and the structure was determined to a resolution of 2.1 Å. The protein folds as a (β/α) 7β-barrel, and a binuclear metal center is found at the C-terminal end of the β-barrel. The purified protein contains a mixture of zinc and iron and is intensely purple at high concentrations. The purple color was determined to be due to a charge transfer complex between iron in the β-metal position and Tyr-97. Mutation of Tyr-97 to phenylalanine or complexation of the metal center with manganese abolished the absorbance in the visible region of the spectrum. Computational docking was used to predict potential substrates for this previously unannotated protein. The enzyme was found to catalyze the hydrolysis of δ- and γ-lactones with an alkyl substitution at the carbon adjacent to the ring oxygen. The best substrate was δ-nonanoic lactone with a k cat/K m of 1.6 × 10 6M -1 s -1. Dr0930 was also found to catalyze the very slow hydrolysis of paraoxon with values of k cat and k cat/K m of 0.07 min -1 and 0.8 M -1 s -1, respectively. The amino acid sequence identity to the phosphotriesterase (PTE) from Pseudomonas diminuta is ∼30%. The eight substrate specificity loops were transplanted from PTE to Dr0930, but no phosphotriesterase activity could be detected in the chimeric PTE-Dr0930 hybrid. Mutation of Phe-26 and Cys-72 in Dr0930 to residues found in the active site of PTE enhanced the kinetic constants for the hydrolysis of paraoxon. The F26G/C72I mutant catalyzed the hydrolysis of paraoxon with a k cat of 1.14 min -1, an increase of 16-fold over the wild-type enzyme. These results support previous proposals that phosphotriesterase activity evolved from an ancestral parent enzyme possessing lactonase activity.

Original languageEnglish (US)
Pages (from-to)2237-2247
Number of pages11
JournalBiochemistry
Volume48
Issue number10
DOIs
StatePublished - Mar 17 2009

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Phosphoric Triester Hydrolases
Deinococcus
Amidohydrolases
Paraoxon
Hydrolysis
Cats
Metals
Lactones
Enzymes
Substrates
Iron
Mutation
Proteins
Manganese
Substrate Specificity
Pseudomonas
Complexation
Phenylalanine
Charge transfer
Zinc

ASJC Scopus subject areas

  • Biochemistry

Cite this

Functional annotation and three-dimensional structure of Dr0930 from deinococcus radiodurans, a close relative of phosphotriesterase in the amidohydrolase superfamily. / Xiang, Dao Feng; Kolb, Peter; Fedorov, Alexander A.; Meier, Monika M.; Fedorov, Lena V.; Nguyen, Tinh T.; Sterner, Reinhard; Almo, Steven C.; Shoichet, Brian K.; Raushel, Frank M.

In: Biochemistry, Vol. 48, No. 10, 17.03.2009, p. 2237-2247.

Research output: Contribution to journalArticle

Xiang, DF, Kolb, P, Fedorov, AA, Meier, MM, Fedorov, LV, Nguyen, TT, Sterner, R, Almo, SC, Shoichet, BK & Raushel, FM 2009, 'Functional annotation and three-dimensional structure of Dr0930 from deinococcus radiodurans, a close relative of phosphotriesterase in the amidohydrolase superfamily', Biochemistry, vol. 48, no. 10, pp. 2237-2247. https://doi.org/10.1021/bi802274f
Xiang, Dao Feng ; Kolb, Peter ; Fedorov, Alexander A. ; Meier, Monika M. ; Fedorov, Lena V. ; Nguyen, Tinh T. ; Sterner, Reinhard ; Almo, Steven C. ; Shoichet, Brian K. ; Raushel, Frank M. / Functional annotation and three-dimensional structure of Dr0930 from deinococcus radiodurans, a close relative of phosphotriesterase in the amidohydrolase superfamily. In: Biochemistry. 2009 ; Vol. 48, No. 10. pp. 2237-2247.
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abstract = "Dr0930, a member of the amidohydrolase superfamily in Deinococcus radiodurans, was cloned, expressed, and purified to homogeneity. The enzyme crystallized in the space group P3 121, and the structure was determined to a resolution of 2.1 {\AA}. The protein folds as a (β/α) 7β-barrel, and a binuclear metal center is found at the C-terminal end of the β-barrel. The purified protein contains a mixture of zinc and iron and is intensely purple at high concentrations. The purple color was determined to be due to a charge transfer complex between iron in the β-metal position and Tyr-97. Mutation of Tyr-97 to phenylalanine or complexation of the metal center with manganese abolished the absorbance in the visible region of the spectrum. Computational docking was used to predict potential substrates for this previously unannotated protein. The enzyme was found to catalyze the hydrolysis of δ- and γ-lactones with an alkyl substitution at the carbon adjacent to the ring oxygen. The best substrate was δ-nonanoic lactone with a k cat/K m of 1.6 × 10 6M -1 s -1. Dr0930 was also found to catalyze the very slow hydrolysis of paraoxon with values of k cat and k cat/K m of 0.07 min -1 and 0.8 M -1 s -1, respectively. The amino acid sequence identity to the phosphotriesterase (PTE) from Pseudomonas diminuta is ∼30{\%}. The eight substrate specificity loops were transplanted from PTE to Dr0930, but no phosphotriesterase activity could be detected in the chimeric PTE-Dr0930 hybrid. Mutation of Phe-26 and Cys-72 in Dr0930 to residues found in the active site of PTE enhanced the kinetic constants for the hydrolysis of paraoxon. The F26G/C72I mutant catalyzed the hydrolysis of paraoxon with a k cat of 1.14 min -1, an increase of 16-fold over the wild-type enzyme. These results support previous proposals that phosphotriesterase activity evolved from an ancestral parent enzyme possessing lactonase activity.",
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T1 - Functional annotation and three-dimensional structure of Dr0930 from deinococcus radiodurans, a close relative of phosphotriesterase in the amidohydrolase superfamily

AU - Xiang, Dao Feng

AU - Kolb, Peter

AU - Fedorov, Alexander A.

AU - Meier, Monika M.

AU - Fedorov, Lena V.

AU - Nguyen, Tinh T.

AU - Sterner, Reinhard

AU - Almo, Steven C.

AU - Shoichet, Brian K.

AU - Raushel, Frank M.

PY - 2009/3/17

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N2 - Dr0930, a member of the amidohydrolase superfamily in Deinococcus radiodurans, was cloned, expressed, and purified to homogeneity. The enzyme crystallized in the space group P3 121, and the structure was determined to a resolution of 2.1 Å. The protein folds as a (β/α) 7β-barrel, and a binuclear metal center is found at the C-terminal end of the β-barrel. The purified protein contains a mixture of zinc and iron and is intensely purple at high concentrations. The purple color was determined to be due to a charge transfer complex between iron in the β-metal position and Tyr-97. Mutation of Tyr-97 to phenylalanine or complexation of the metal center with manganese abolished the absorbance in the visible region of the spectrum. Computational docking was used to predict potential substrates for this previously unannotated protein. The enzyme was found to catalyze the hydrolysis of δ- and γ-lactones with an alkyl substitution at the carbon adjacent to the ring oxygen. The best substrate was δ-nonanoic lactone with a k cat/K m of 1.6 × 10 6M -1 s -1. Dr0930 was also found to catalyze the very slow hydrolysis of paraoxon with values of k cat and k cat/K m of 0.07 min -1 and 0.8 M -1 s -1, respectively. The amino acid sequence identity to the phosphotriesterase (PTE) from Pseudomonas diminuta is ∼30%. The eight substrate specificity loops were transplanted from PTE to Dr0930, but no phosphotriesterase activity could be detected in the chimeric PTE-Dr0930 hybrid. Mutation of Phe-26 and Cys-72 in Dr0930 to residues found in the active site of PTE enhanced the kinetic constants for the hydrolysis of paraoxon. The F26G/C72I mutant catalyzed the hydrolysis of paraoxon with a k cat of 1.14 min -1, an increase of 16-fold over the wild-type enzyme. These results support previous proposals that phosphotriesterase activity evolved from an ancestral parent enzyme possessing lactonase activity.

AB - Dr0930, a member of the amidohydrolase superfamily in Deinococcus radiodurans, was cloned, expressed, and purified to homogeneity. The enzyme crystallized in the space group P3 121, and the structure was determined to a resolution of 2.1 Å. The protein folds as a (β/α) 7β-barrel, and a binuclear metal center is found at the C-terminal end of the β-barrel. The purified protein contains a mixture of zinc and iron and is intensely purple at high concentrations. The purple color was determined to be due to a charge transfer complex between iron in the β-metal position and Tyr-97. Mutation of Tyr-97 to phenylalanine or complexation of the metal center with manganese abolished the absorbance in the visible region of the spectrum. Computational docking was used to predict potential substrates for this previously unannotated protein. The enzyme was found to catalyze the hydrolysis of δ- and γ-lactones with an alkyl substitution at the carbon adjacent to the ring oxygen. The best substrate was δ-nonanoic lactone with a k cat/K m of 1.6 × 10 6M -1 s -1. Dr0930 was also found to catalyze the very slow hydrolysis of paraoxon with values of k cat and k cat/K m of 0.07 min -1 and 0.8 M -1 s -1, respectively. The amino acid sequence identity to the phosphotriesterase (PTE) from Pseudomonas diminuta is ∼30%. The eight substrate specificity loops were transplanted from PTE to Dr0930, but no phosphotriesterase activity could be detected in the chimeric PTE-Dr0930 hybrid. Mutation of Phe-26 and Cys-72 in Dr0930 to residues found in the active site of PTE enhanced the kinetic constants for the hydrolysis of paraoxon. The F26G/C72I mutant catalyzed the hydrolysis of paraoxon with a k cat of 1.14 min -1, an increase of 16-fold over the wild-type enzyme. These results support previous proposals that phosphotriesterase activity evolved from an ancestral parent enzyme possessing lactonase activity.

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